Barley gene for thioredoxin and NADP-thioredoxin reductase

ABSTRACT

The present invention provides barley thioredoxin h nucleic acids and NADP-thioredoxin reductase nucleic acids, the respective encoded proteins and methods of use.

This application is a Divisional application Ser. No. 09/540,014 filedMar. 31, 2000 now U.S. Pat. No. 6,380,372, which claims the benefit ofthe filing date of application Ser. No. 60/127,198, filed Mar. 31, 1999;application Ser. No. 60/169,162 filed Dec. 6, 1999; application Ser. No.60/177,740 filed Jan. 21, 2000; and application Ser. No. 60/177,739filed Jan. 21, 2000, all of which are expressly incorporated byreference in their entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant 9803835 fromthe U.S. Department of Agriculture. The Government has certain rights tothis invention.

BACKGROUND OF THE INVENTION

Thioredoxins are small (about 12 kDa) thermostable proteins withcatalytically active disulfide groups. This class of proteins has beenfound in virtually all organisms, and has been implicated in myriadbiochemical pathways (Buchanan et al., 1994). The active site ofthioredoxin has two redox-active cysteine residues in a highly conservedamino acid sequence; when oxidized, these cysteines form a disulfidebridge (—S—S—) that can be reduced to the sulfhydryl (—SH) level througha variety of specific reactions. In physiological systems, thisreduction may be accomplished by reduced ferredoxin, NADPH, or otherassociated thioredoxin-reducing agents. The reduced form of thioredoxinis an excellent catalyst for the reduction of even the most intractabledisulfide bonds.

Generally only one kind of thioredoxin is found in bacterial or animalcells. In contrast, photosynthetic organisms have three distinct typesof thioredoxin. Chloroplasts contain a ferredoxin/thioredoxin systemcomprised of ferredoxin, ferredoxin-thioredoxin reductase andthioredoxins f and m, which function in the light regulation ofphotosynthetic enzymes (Buchanan, 1991; Scheibe, 1991; Vogt et al,1986). The other thioredoxin enzyme system is analogous to thatestablished for animals and most microorganisms, in which thioredoxin(h-type in plants) is reduced by NADPH and NADPH-thioredoxin reductase(NTR) (Johnson et al., 1987a; Florencio et al., 1988; Suske et al.,1979). The reduction of thioredoxin h by this system can be illustratedby the following equation:

Some plant species contain a family of closely related thioredoxin hproteins, which probably perform different physiological functions.Specific plants in which multiple thioredoxin h proteins have been foundinclude spinach (Florencio et al., 1988), wheat (Johnson et al., 1987),rice (Ishiwatari et al., 1995), and Arabidopsis (Rivera-Madrid et al.,1995). The type-h thioredoxin was discovered considerably after thediscovery of the m and f types, and because of this much less is knownabout this cytosolic thioredoxin and its physiological functions.Considerable work is currently directed toward studying thioredoxin hproteins (Besse and Buchanan, 1997).

Thioredoxin h is widely distributed in plant tissues and exists inmitochondria, endoplasmic reticulum (ER) and the cytosol(Bodenstein-Lang et al., 1989; Marcus et al., 1991; Vogt et al. 1986).Plant thioredoxin h is involved in a wide variety of biologicalfunctions. Thioredoxin h functions in the reduction of intramoleculardisulfide bridges of a variety of low molecular-weight, cystine-richproteins, including thionins (Johnson et al., 1987b), proteaseinhibitors and chloroform/methanol-soluble proteins (CMproteins)(Kobrehel et al., 1991). It is likely that cytoplasmicthioredoxins participate in developmental processes: for examplethioredoxin h has been shown to function as a signal to enhancemetabolic processes during germination and seedling development(Kobrehel et al., 1992; Lozano et al., 1996; Besse et al., 1996).Thioredoxin h has also been demonstrated to be involved inself-incompatibility in Phalaris coerulescens (Li et al., 1995) andBrassica napus (Bower et al., 1996). Several functions have beenhypothesized for rice thioredoxin h, which is believed to be involved intranslocation in sieve tubes (Ishiwatari et al., 1995).

Uses of thioredoxin include incorporation into hair care products (U.S.Pat. No. 4,935,231) and neutralization of certain venoms and toxins (seeU.S. Pat. No. 5,792,506). Recent research into thioredoxin activity hasalso focused on harnessing the reducing power of this protein for foodtechnology. For example, U.S. Pat. No. 5,792,506 to Buchanan(Neutralization of Food Allergens by Thioredoxin), and Buchanan et at.(1998) describe the use of thioredoxin to reduce the allergenicity offoods through thioredoxin-medicated reduction of intramoleculardisulfide bonds found in various allergenic food proteins (e.g., inmilk, soya and wheat proteins) (Buchanan et al., 1997; del Val et al.,1999). In addition, it has been shown that reduction of disulfideprotein allergens in wheat and milk by thioredoxin decreases theirallergenicity (Buchanan et al., 1997; del Val et al., 1999). Thioredoxintreatment also increases the digestibility of the major allergen of milk(β-lactoglobulin)(del Val at al., 1999), as well as other disulfideproteins (Lozano et al., 1994; Jiao et al., 1992).

Thioredoxin h has been shown to be useful as a food additive to enhancethe baking qualities of cereal flour (Bright et al., 1983). For example,improvement in dough strength and bread quality properties ofpoor-quality wheat flour results from the addition of thioredoxin (Wonget al., 1993; Kobrehel et al., 1994). This has been attributable to thethioredoxin-catalyzed reduction of intramolecular disulfide bonds in theflour proteins, specifically the glutenins, resulting in the formationof new intermolecular disulfide bonds (Besse and Buchanan, 1997). Thus,the addition of exogenous thioredoxin promotes the formation of aprotein network that produces flour with enhanced baking quality.Kobrehel et al., (1994) have observed that the addition of thioredoxin hto flour of non-glutenous cereals such as rice, maize and sorghumpromotes the formation of a dough-like product. Hence, the addition ofexogenous thioredoxin may be used to produce baking dough fromnon-glutenous cereals.

cDNA clones encoding thioredoxin h have been isolated from a number ofplant species, including Arabidopsis thaliana (Rivera-Madrid et al.,1993; Rivera-Madrid et al., 1995), Nicotiana tabacum (Marty and Meyer,1991; Brugidou et al., 1993), Oryza sativa (Ishiwatari et al., 1995),Brassica nepus (Bower et al., 1996), Glycine max (Shi and Bhattacharyya,19986), and Triticum aestivum (Gautier et al., 1998).

Thioredoxin and NTR were first characterized in Escherichia coli as thehydrogen donor system for ribonucleotide reductase (Laurent et al.,1964; Moore et al., 1964) The E. coli NTR gene has been isolated (Russeland Model, 1988) and the three-dimensional structure of the protein hasbeen analyzed (Kuriyan et al., 1991). Some other NTR genes have beenisolated and sequenced from bacteria, fungi and mammals. Recently,Jacquot et al. (1994) have reported a successful isolation andsequencing of two cDNAs encoding the plant Arabidopsis thaliana NTRs.The subsequent expression of the recombinant A. thaliana NTR protein inE. coli cells (Jacquot et al., 1994) and its first eukaryotic structure(Dai et al., 1996) have also been reported.

Thioredoxin and NTR were first characterized in Escherichia coli as thehydrogen donor system for ribonucleotide reductase (Laurent et al.,1964; Moore et al., 1964) The E. coli NTR gene has been isolated (Russeland Model, 1988) and the three-dimensional structure of the protein hasbeen analyzed (Kuriyan et al., 1991). Some other NTR genes have beenisolated and sequenced from bacteria, fungi, and mammals. Recently,Jacquot et al., (1994) have reported a successful isolation andsequencing of two cDNAs encoding the plant Arabidopsis thaliana NTRs.The subsequent expression of the recombinant A. thaliana NTR protein inE. coli cells (Jacquot et al., 1994) and its first eukaryotic structure(Dai et al., 1996) have also been reported.

Here we report isolated nucleic acids encoding the barley genes forthioredoxin h and NADP-thioredoxin reductase; isolated barleythioredoxin h and NADP-thioredoxin reductase proteins, and methods ofuse.

SUMMARY OF THE INVENTION

The invention provides isolated nucleic acids encoding barleythioredoxin and NADP-thioredoxin reductase proteins and methods of use.

In other aspect the invention provides expression vectors comprisingnucleic acids encoding barley thioredoxin and NADP-thioredoxin andtransformed host cells. Accordingly, the invention provides methods ofexpressing an isolated barley thioredoxin and NADP-thioredoxin reductasepolypeptides.

In a further aspect the invention provides transgenic plants comprisingthe expression vectors. In a preferred embodiment, the transgenic plantsoverexpress barley thioredoxin and NADP-thioredoxin reductasepolypeptides. The polypeptides of the invention, expressed in atransgenic plant either alone or in combination alters the redox statusof a plant in comparison to a nontransgenic plant of the same species.In a preferred embodiment, the expressed polypeptide transgene altersthe redox status of a seed or grain, thereby altering the biochemicaland biological properties of a seed or grain. The seed or grain providesadvantages in increased germination efficiency, decreased allergenicity,increased protein solubility, increased digestibility.

In another aspect, the invention provides methods of expressing a barleythioredoxin or NADP-thioredoxin reductase polypeptides.

In yet another aspect, the invention provides of expressing a barleythioredoxin or NADP-thioredoxin reductase polypeptide. Accordingly, theinvention provides isolated barley thioredoxin or NADP-thioredoxinreductase polypeptides.

In still yet another aspect the invention provides methods ofidentifying a bioactive agent that binds and preferably reduces abiological activity of a barley thioredoxin or NADP-thioredoxinreductase polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of the amino acid sequences of barleythioredoxin h and two wheat thioredoxin h proteins (SEQ ID NO:2, 4 and6) respectively).

FIG. 2 shows a comparison of the nucleic acid sequence encoding barleythioredoxin h and two wheat thioredoxin h molecules, and a consensussequence derived by the comparison of these three sequences. (SEQ IDNOS:1, 3 and 5 respectively). The BTRXh and wheat Trxh have about 90%sequence identity from positions 30-394.

FIG. 3 shows the positions of primers used for PCR amplification toisolate H. vulgare NADP-thioredoxin reductase (NTR).

FIGS. 4A-C shows the deduced amino acid sequence of H. vulgareNADP-thioredoxin reductase (NTR) and homologies. Panel A shows the aminoacid sequence alignment of H. vulgare NADP-thioredoxin reductase (NTR)and the NTR sequences of A. thaliana (SEQ ID NO:24) and E. coli (SEQ IDNO:25). Amino acid identities are shown by shaded residues. Panel Bshows the percent similarity and percent divergence between amino acidsequences of H. vulgare NTR and the NTR sequences of A. thaliana and E.coli. Panel C shows the phylogenetic tree (in relative units) of H.vulgare NTR and related sequences from A. thaliana and E. coli.

FIGS. 5A-D shows the nucleotide sequence of H. vulgare NADP-thioredoxinreductase (NTR) gene (SEQ ID NO:10) and homolgoies. Panel A shows thenucleotide sequence alignment of H. vulgare NTR gene and the NTRsequences of A. thaliana and E. coli. Nucleotides conserved in at leasttwo out of three different NTR genes are shaded. Panel B shows thenucleotide sequence alignment of H. vulgare NTR gene and the NTRsequences of A. thaliana (SEQ ID NO:26) and E. coli (SEQ ID NO:27).Nucleotides conserved nucleotides the three different NTR genes areshaded. Panel C shows the percent homology and percent divergencebetween nucleotide sequences of H. vulgare NTR and the NTR sequences ofA. thaliana and E. coli. Panel D shows that the phylogenetic tree (inrelative units) of H. vulgare NTR and related nucleotide sequences fromA. thaliana and E. coli.

FIG. 6 shows the thioredoxin h constructs used for transformation.

FIG. 7 shows the thioredoxin activity profile of various barley grainstransformed with wheat thioredoxin gene (wtrxh).

FIG. 8 shows the effects of heat treatment on thioredoxin activity ofcrude extracts from barley grains.

FIGS. 9A-B shows a western blot analysis of extract from segregating T₁barley grain of stable transformants containing wtrxh. Panel A: lanes 1and 6, control barely extract (cv. Golden Promise); lane 2, bread wheatextract (Triticum aestivum, cv. Capitole); lane 3, extract from GPdBhssBarWtrx 22; lane 4, extract from GPdBhssBarWtrx 29; lane 5, extract fromGPdBhBarWtrx 2. Panel B: lane 1, GPdBhBaarWtrx 2; lane 2 control barleyextract. W, wheat; B, barley.

FIG. 10 shows western blot analysis of extracts of T₁, T₂ and T₃ barleygrain transformed with wtrxh. Forty micrograms of soluble proteinsextracted from 10-20 grains of each line were fractionated by SDS/PAGE.Lane 1, wheat germ thioredoxin h; lane 2, nontransgenic control ofGP4-96; lane 3, null segregant T₂ grain of GPdBhssBarWtrx-29-11-10; lane4, heterozygous T₂ grain of GPdBhssWtrx-29; lane 5, homozygous T₂ grainof GPdBhssBarWtrx-29-3; lane 6, homozygous T₂ grain ofGPdBhssBarWtrx-29-3-2; lane 7, prestained standards (aprotinin, 0.9 kDa;lysozyme, 17.8 kDa; soybean trypsin inhibitor, 30.6 kDa; carbonicanhydrase, 41.8 kDa; BSA, 71 kDa).

FIG. 11 shows the nucleic acid sequence of the B₁-hordein promoter (SEQID NO: 11) and the 57 base pair B₁-hordein signal sequence (underlined).FIG. 12 shows the nucleic acid sequence of the D -hordein promoter (SEQID NO: 12) and the 63 base pair D-hordein signal sequence (underlined).

FIGS. 13A-C shows the effect of overexpressed thioredoxin h onpullulanase activity in transgenic barley grain during germination andseedling development. A homozygous line, GPdBhssBarWtrx-29-3, and a nullsegregant, GPdBhssBarWtrx-29-11-10, were used for the pullulanaseassays. Pane A: Pullulanase was assayed spectrophotometrically bymeasuring the dye released from red pullulan substrate at 534 nm. PanelB: Pullulanase was separated on native 7.5% polyacrylamide gelscontaining the red pullulan substrate. Activity, identified bycomparison with purified barley pullulanase, is seen as clear areas thatdeveloped on incubating the gel in 0.2 M succinate buffer, pH 6.0, for 1hr at 37° C. Panel C: The gel in Panel B was scanned and analyzed byintegration of the activity bands.

FIGS. 14A-D shows the change in the activity and abundance of amylasesin transgenic and null segregant barley grains during germination andseedling development based on an activity gel. Panel A: abundance ofalpha-amylases in null segregant based on western blot Panel B: Totalamylase activity in null segregant. Panel C: abundance of alpha-amylasesin thioredoxin overexpressing grains. Panel D: total amylase activity inthioredoxin overexpressed grains.

FIG. 15 shows the effect of overexpressed thioredoxin h on the activityof the major form of alpha-amylase during germination and seedingdevelopment. The size of the major alpha-amylase activity band in FIG.14 was estimated by its rate of mobility during eletrophoresis.

FIGS. 16A-B shows the effect of overexpressed thioredoxin h on theabundance of alpha-amylase A and B isozymes during germination andseedling development. The figure represents western blots of IEF gelsdeveloped for the null segregant and transgenic barley grains. Panel A:Null segregant. Panel B: Transgenic with thioredoxin overexpressed.

FIG. 17 shows the effect of overexpressed wheat thioredoxin h on thegermination of null segregant and transgenic (homozygous) barley grains.

FIG. 18 shows the relative redox status of protein fractions intransgenic barley grain overexpressing wheat thioredoxin h in comparisonto the null segregant in dry and germination grain.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting (SEQ ID NO:1-24) are shown using standard letter abbreviationsfor nucleotide bases, and three letter code for amino acids. Only onestrand of each nucleic acid sequence is shown, but it is understood thatthe complementary strand is included by any reference to the displayedstrand.

SEQ ID NO:1 shows the nucleic acid sequence of the barley thioredoxin hcDNA.

SEQ ID NO:2 shows the amino acid sequence of the barley thioredoxin hprotein.

SEQ ID NO:3 shows the nucleic acid sequence of a wheat thioredoxin hcDNA, GenBank accession number X699 15.

SEQ ID NO:4 shows the amino acid sequence of a wheat thioredoxin hprotein.

SEQ ID NO:5 shows the nucleic acid sequence of a wheat thioredoxin hcDNA, GenBank accession number AJ00 1903.

SEQ ID NO:6 shows the amino acid sequence of a wheat thioredoxin hprotein.

SEQ ID NO:11 shows the nucleic acid sequence of the barley B₁-hordeinpromoter and signal sequence.

SEQ ID NO:12 shows the nucleic acid sequence of the barley D-hordeinpromoter and signal sequence.

Other SEQ ID NOs: are described herein.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Lewin, Genes V published by Oxford University Press, 1994(ISBN 0-19-854287-9); Kendrew et al (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology, a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8); Ausubel et al. (1987)Current Protocols in Molecular Biology, Green Publishing; Sambrook etal. (989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y.).

In order to facilitate review of the various embodiments of theinvention, the following definitions are provided:

Thioredoxin protein: A large number of plant, animal, and microbialthioredoxin proteins have been characterized, and the genes encodingmany of these proteins have been cloned and sequenced. The presentinvention is preferably directed to the use of thioredoxin h proteins,although other thioredoxin proteins may also be employed to producetransgenic plants as described herein. Among the thioredoxin h proteinsfrom plants that have been described to date are thioredoxin h proteinsfrom Arabidopsis thaliana (Rivera-Madrid et al., 1993; Rivera-Madrid etal., 1995), Nicotiana tabacum (Marty and Meyer, 1991; Brugidou et al.,1993), Oryza sativa (Ishiwatari et al., 1995), Brassica napus (Bower etal., 1996), Glycine max (Shi and Bhattacharyya, 1996), and Triticumaestivum (Gautier et al., 1998). The amino acid sequences of these andother thioredoxin h proteins, and the nucleotide sequence of cDNAsand/or genes that encode these proteins, are available in the scientificliterature and publicly accessible sequence databases. For example, acDNA encoding thioredoxin h from Picea mariana is described in accessionnumber AF051206 (NID g2982246) of GenBank, and located by a search usingthe Entrez browser/nucleotide sequence search of the National Center forBiotechnology Information website, www.ncbi.nim.nih.gov. The cDNAencoding the Triticum aestivum thioredoxin h protein used in theExamples described below is described on the same database underaccession number X69915 (NID g2995377).

The present invention may be practiced using nucleic acid sequences thatencode full length thioredoxin h proteins, as well as thioredoxin hderived proteins that retain thioredoxin h activity. Thioredoxin hderived proteins which retain thioredoxin biological activity includefragments of thioredoxin h, generated either by chemical (e.g.enzymatic) digestion or genetic engineering means; chemicallyfunctionalized proteins molecules obtained starting with the exemplifiedprotein or nucleic acid sequences, and protein sequence variants. Thus,the term “thioredoxin h protein” encompasses full length thioredoxin hproteins, as well as such thioredoxin h derived proteins that retainthioredoxin h activity.

Thioredoxin protein may be qualified in biological samples (such asseeds) either in terms of protein level, or in terms of thioredoxinactivity. Thioredoxin protein level may be determined using a westernblot analysis followed by quantitative scanning of the image asdescribed in detail below. Thioredoxin activity may be quantified usinga number of different methods known in the art. Preferred methods ofmeasuring thioredoxin biological activity attributable to thioredoxin hin plant extracts include NADP/malate dehydrogenase activation (Johnsonet al., 1987a,b) and reduction of 2′,5′-dithiobis(2-nitrobenzoic acid)(DTNB) via NADP-thioredoxin reductase (Florencio et al., 1988; U.S. Pat.No. 5,792,506). Due to the potential for interference fromnon-thioredoxin h enzymes that use NADPH, accurate determination ofthioredoxin h activity should preferably be made using partiallypurified plant extracts. Standard protein purification methods (e.g.(NH₄)₂SO₄ extraction) can be used to accomplish this partialpurification. The activity of thioredoxin h may also be expressed interms of specific activity, i.e., thioredoxin activity per unit ofprotein present, as described in more detail below.

Probes and primers: Nucleic acid probes and primers may readily beprepared based on the nucleic acids provided by this invention. A probecomprises an isolated nucleic acid attached to a detectable label orreporter molecule. Typical labels include radioactive isotopes, ligands,chemiluminescent agents, and enzymes. Methods for labeling and guidancein the choice of labels appropriate for various purposes are discussed,e.g., in Sambrook et al. (1989) and Ausubel et al. (1987).

Primers are short nucleic acids, preferably DNA oligonucleotides about15 nucleotides or more in length. Primers may be annealed to acomplementary target DNA strand by nucleic acid hybridization to formhybrid between the primer and the target DNA strand, and then extendedalong the target DNA strand by a DNA polymerase enzyme. Primer pairs canbe used for amplification of a nucleic acid sequence, e.g, by thepolymerase chain reaction (PCR) or other nucleic-acid amplificationmethods known in the art.

Methods for preparing and using probes and primers are described, forexample, in Sambrook et al. (1989), Ausubel et al. (1987), and Innis etal., (1990). PCR primer pairs can be derived from a known sequence, forexample, by using computer programs intended for that purpose such asPrimer (Version 0.5, © 1991, Whitehead Institute for BiomedicalResearch, Cambridge, Mass.). One of skill in the art will appreciatethat the specificity of a particular probe or primer increases with itslength. Thus, for example, a primer comprising 20 consecutivenucleotides of the barley thioredoxin h cDNA will anneal to a targetsequence such as a thioredoxin h homologue from a different barleycultivar contained within a cDNA or genomic DNA library with a higherspecificity than a corresponding primer of only 15 nucleotides. Thus, inorder to obtain greater specificity, probes and primers may be selectedthat comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotide ofthe barley thioredoxin h cDNA or gene sequences.

Accordingly, oligonucleotides that are derived from the barleythioredoxin h and NTR nucleic acids are encompassed within the scope ofthe present invention. Preferably, such oligonucleotide primers willcomprise a sequence of at least 15-20 consecutive nucleotides of thebarley thioredoxin h encoding sequences.

Promoter: A regulatory nucleic acid sequence, typically located upstream(5′) of a gene that, in conjunction with various cellular proteins, isresponsible for regulating the expression of the gene. Promoters mayregulate gene expression in a number of ways. For example, theexpression may be tissue-specific, meaning that the gene is expressed atenhanced levels in certain tissues, or developmentally regulated, suchthat the gene is expressed at enhanced levels at certain times duringdevelopment, or both.

In a preferred embodiment, a transgene of the invention is expressed inan edible part of a plant. By “edible” herein is meant at least a partof a plant that is suitable for consumption by humans or animals (fish,crustaceans, isopods, decapods, monkeys, cows, goats, pigs, rabbits,horses, birds (chickens, parrots etc). Accordingly, “edible” embracesfood for human consumption and feed for animal consumption and includes,for example, dough, bread, cookies, pasta, pastry, beverages, beer, foodadditives, thickeners, malt, extracts made from an edible part ofplants, animals feeds, and the like. An edible part of a plant includesfor example, a root, a tuber, a seed, grain, a flower, fruit, leaf etc.The skilled artisan is aware that expression of the transgene iseffected in any tissue, organ or part of a plant by employing a promoterthat is active in the selected part of the plant the transgene is to beexpressed. In a preferred embodiment the transgene is expressed in aseed, preferably under control of a seed- or grain-specific promoter.

The expression of a transgene in seeds or grains according to thepresent invention is preferably accomplished by operably linking aseed-specific or grain-specific promoter to the nucleic acid moleculeencoding the transgene protein. In this context, “seed-specific”indicates that the promoter has enhanced activity in seeds compared toother plant tissues; it does not require that the promoter is solelyactive in the seeds. Accordingly, “grain-specific” indicates that thepromoter has enhanced activity in grains compared to other planttissues; it does not require that the promoter is solely active in thegrain. Preferably, the seed- or grain-specific promoter selected will,at the time when the promoter is most active in seeds, produceexpression of a protein in the seed of a plant that is at least abouttwo-fold greater than expression of the protein produced by that samepromoter in the leaves or roots of the plant. However, given the natureof the thioredoxin protein, it may be advantageous to select a seed- orgrain-specific promoter that causes little or no protein expression intissues other than seed or grain. In a embodiment, a promoter isspecific for seed and grain expression, such that, expression in theseed and grain is enhanced as compared to other plant tissues but doesnot require that the promoter be solely activity in the grain and seed.In a preferred embodiment, the promoter is “specific” for a structure orelement of a seed or grain, such as an embryo-specific promoter. Inaccordance with the definitions provided above, an embryo-specificpromoter has enhanced activity. In an embryo as compared to other partsof a seed or grain or a plant and does not require its activity to belimited to an embryo. In a preferred embodiment, the promoter is“maturation-specific” and accordingly has enhanced activitydevelopmentally during the maturation of a part of a plant as comparedto other parts of a plant and does not require is activity to be limitedto the development of a part of a plant.

A seed- or grain-specific promoter may produce expression in varioustissues of the seed, including the endosperm, embryo, and aleurone orgrain. Any seed- or grain-specific promoter may be used for thispurpose, although it will be advantageous to select a seed- orgrain-specific promoter that produces high level expression of theprotein in the plant seed or grain. Known seed- or grain-specificpromoters include those associated with genes that encode plant seedstorage proteins such as genes encoding; barley hordeins, riceglutelins, oryzins, or prolamines; wheat gliadins or glutenins; maizezeins or glutelins; maize embryo-specific promoter; oat glutelins;sorghum kafirins; millet pennisetins; or rye secalins.

The barley hordein promoters (described in more detail below) are seed-or grain-specific promoters that were used in the illustrative Examples(Cameron-Mills, 1980; Cameron-Mills et al., 1980, 1988a,b).

In certain embodiments, the seed- or grain-specific promoter that isselected is a maturation-specific promoter. The use of promoters thatconfer enhanced expression during seed or grain maturation (such as thebarley hordein promoters) may result in even higher levels ofthioredoxin expression in the seed.

By “seed or grain-maturation” herein refers to the period starting withfertilization in which metabolizable food reserves (e.g., proteins,lipids, starch, etc.) are deposited in the developing seed, particularlyin storage organs of the seed, including the endosperm, testa, aleuronelayer, embryo, and scutellar epithelium, resulting in enlargement andfilling of the seed and ending with seed desiccation.

Members of the grass family, which include the cereal grains, producedry, one-seeded fruits. This type of fruit, is strictly speaking, acaryopsis but is commonly called a kernel or grain. The caryopsis of afruit coat or pericarp, which surrounds the seed and adhere tightly to aseed coat. The seed consist of an embryo or germ and an endospermenclosed by a nucellar epidermis and a seed coat. Accordingly the graincomprises the seed and its coat or pericarp. The seed comprises theembryo and the endosperm. (R. Carl Hoseney in “Principles of CerealScience and Technology”, expressly incorporated by reference in itsentirety).

Starch: A poylsaccharide made up of a chain of glucose units joined byalpha-1,4 linkages, either unbranched (amylose) or branched(amylopectin) at alpha-1,6-linkages.

Dextran: Any of a variety of storage poylsaccharides, usually branched,made of glucose residues joined by alpha-1,6-linkages.

Dextrin or Limit Dextrin: Any of a group of small solublepolysaccharides, partial hydrolysis products of starch, usually enrichedin alpha-1,6-linkages.

Germination: A resumption of growth of a plant embryo in favorableconditions after seed maturation and drying (dessication), and emergenceof young shoot and root from the seed.

Allergen: An antigenic substance that induces an allergic reaction in asusceptible host. Accordingly, a susceptible host has an immune status(hypersensitivity) that results in an abnormal or harmful immunereaction upon exposure to an allergen. In a preferred embodiment, thetransgenic grains of the invention have reduced allergenicity incomparison to nontransgenic grains. The immune reaction can be immediateor delayed; cell mediated or antibody mediated; or a combinationthereof. In a preferred embodiment, the allergic reaction is animmediate type hypersensitivity.

Digestion: By “digestion” herein is meant the conversion of a moleculeor compound to one or more of its components. Accordingly,“digestibility” relates to the rate and efficiency at which theconversion to one or more of its components occurs. In a preferembodiment a “digestible compound” is, for example, a food, that isconverted to its chemical components by chemical or enzymatic means. Forexample, dextran is converted to dextrin, polysaccharide,monosaccharides, limit dextrin etc; a protein is converted to apolypeptides, oligopeptides, amino acids, ammonia etc.; a nucleic acidis converted to oligonucleotides, nucleotides, nucleosides, purine,pyrimidines, phosphates etc. In a preferred embodiment, the transgenicgrains of the invention have increased digestibility, i.e. are moreefficiently or rapidly digested in comparison to nontransgenic grain.

Germination: A resumption of growth of a plant embryo in favorableconditions after seed or grain maturation and drying (dessication), andemergence of young shoot and root from the seed or grain.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include one or morenucleic add sequences that permit it to replicate in one or more hostcells, such as origin(s) of replication. A vector may also include oneor more selectable marker genes and other genetic elements known in theart.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, plant or animalcell, including transfection with viral vectors, transformation byAgrobacterium, with plasmid vectors, and introduction of naked DNA byelectroporation, lipofection, and particle gun acceleration and includestransient as well as stable transformants.

Isolated: An “isolated” biologic component (such as a nucleic acid orprotein or organelle) has been substantially separated or purified awayfrom other biological components in the cell or the organism in whichthe component naturally occurs, i.e., other chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term embracesnucleic acids including chemically synthesized nucleic acids and alsoembraces proteins prepared by recombinant expression in vitro or in ahost cell and recombinant nucleic acids as defined below

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary, join two protein-coding regions in the samereading frame. With respect to polypeptides, two polypeptide sequencesmay be operably linked by covalent linkage, such as through peptidebonds or disulfide bonds.

Recombinant: By “recombinant nucleic acid” herein is meant a nucleicacid that has a sequence that is not naturally occurring or has asequence that is made by an artificial combination of two otherwiseseparated segments of sequence. This artificial combination is oftenaccomplished by chemical synthesis or, more commonly, by the artificialmanipulation of nucleic acids, e.g., by genetic engineering techniques,such as by the manipulation of at least one nucleic acid by arestriction enzyme, ligase, recombinase, and/or a polymerase. Onceintroduced into a host cell, a recombinant nucleic acid is replicated bythe host cell, however, the recombinant nucleic acid once replicated inthe cell remains a recombinant nucleic acid for purposes of thisinvention. By “recombinant protein” herein is meant a protein producedby a method employing a recombinant nucleic acid. As outlined above“recombinant nucleic acids” and “recombinant proteins” also are“isolated”, as described above.

Complementary DNA (cDNA): A piece of DNA that is synthesized in thelaboratory by reverse transcription of an RNA, preferably an RNAextracted from cells. cDNA produced from mRNA typically lacks internal,non-coding segments (introns) and regulatory sequences that determinetranscription.

Open reading frame (ORF): A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into a peptide.

Transgenic plant: As used herein, this term refers to a plant thatcontains recombinant or isolated genetic material not normally found inplants of this type and which has been introduced into the plant inquestion (or into progenitors of the plant) by human manipulation. Thus,a plant that is grown from a plant cell into which recombinant DNA isintroduced by transformation is a transgenic plant, as are all offspringof that plant that contain the introduced transgene (whether producedsexually or asexually). It is understood that the term transgenic plantencompasses the entire plant and parts of said plant, for instancegrains, seeds, flowers, leaves, roots, fruit, pollen, stems etc.

The present invention is applicable to both dicotyledonous plants (e.g.tomato, potato, soybean, cotton, tobacco, etc.) and monocotyledonousplants, including, but not limited to graminaceous monocots such aswheat (Triticum spp.), rice (Oryza spp.), barley (Hordeum spp.), oat(Avene spp.), rye (Secale spp.), corn (Zea mays), sorghum (Sorghum spp.)and millet (Pennisetum spp). For example, the present invention can beemployed with barley genotypes including, but not limited to Morex,Harrington, Crystal Stander, Moravian III, Galena, Salome, Steptoe,Klages, Baronesse, and with wheat genotypes including, but not limitedto Yecora Rojo, Bobwhite, Karl and Anza. In general, the invention isparticularity useful in cereals.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified barleythioredoxin h protein preparation is one in which the barley thioredoxinh protein is more enriched or more biochemically active or more easilydetected than the protein is in its natural environment within a cell orplant tissue. Accordingly, “purified” embraces or includes the removalor inactivation of an inhibitor of a molecule of interest. In apreferred embodiment a preparation of barley thioredoxin h protein ispurified such that the barley thioredoxin h represents at least 5-10% ofthe total protein content of the preparation. For particularapplications, higher protein purity may be desired, such thatpreparations in which barley thioredoxin h represents at least 50% or atleast 75% or at least 90% of the total protein content may be employed.

Ortholog: Two nucleotide or amino acid sequences are orthologs of eachother if they share a common ancestral sequence and diverged when aspecies carrying that ancestral sequence split into two species,sub-species, or cultivars. Orthologous sequences are also homologoussequences.

II. Thioredoxin h (BTRXh) and NADP-thioredoxin Reductase (NTR) fromBarley (Hordeum vulgare L.)

Herein are provided BTRXh and NTR proteins and nucleic acids whichencode such proteins. Also provided are methods of screening for abioactive agent capable of binding and preferably modulating theactivity of the BTRXh or NTR protein. The method comprises combining aBTRXh or an NTR protein and a candidate bioactive agent and a cell or apopulation of cells, and determining the effect on the cell in thepresence and absence of the candidate agent. Other screening assaysincluding binding assays are also provided herein as described below.

NTR belongs to the pyridine nuclieotide-disulfide oxidoreductase family(Pai, 1991), which includes glutathione reductase, lipoamidedehydrogenase, mercuric reductase and trypanothionine reductase, whichcatalyze the transfer of electrons from a pyridine nucleotide via aflavin carrier to, in most cases, disulfide-containing substrates.Preferably, NTR is barley H. vulgare NTR and is a flavoenzyme thatreduces thioredoxin h using NADPH. We have found that barley NTR reduceswheat thioredoxin h (Cho et al. 1999 (PNAS)).

A barley thioredoxin protein is a barley protein having thioredoxinbiological activity. Plant thioredoxins are generally categorized intothree subgroups (m, f, and h) based on subcellular localization andspecificity of enzyme activation. A barley thioredoxin h (BTRXh) proteinis a barley protein having thioredoxin protein biological activity andsharing amino acid sequence identity and/or is encoded by a nucleic acidthat hybridizes under high stringency conditions to the exemplifiedBTRXh nucleic acid as described below. Thioredoxin proteins typicallycontain a consensus active site—WCGPC (residues 45-49 of SEQ ID NO:2).Though it is not absolutely required, in general thioredoxin proteinscan also be identified by the presence of this or a similar sequence.

A BTRXh and an NTR protein of the present invention also may beidentified in alternative ways. “Protein” in this sense includesproteins, polypeptides, and peptides.

The BTRXh and NTR proteins of the invention fall into two generalclasses: proteins that are completely novel, i.e. are not part of apublic database as of the time of discovery, although they may havehomology to either known proteins or peptides encoded by expressedsequence tags (ESTs) and the like. Alternatively, the BTRXh and NTRproteins are known proteins, but that were not known to be,respectively, thioredoxins or oxidoreductase that preferably reducethioredoxin h. Accordingly, a NTR protein may be initially identified byits association with a protein known to be involved in the reduction ofthioredoxin. A BTRXh protein may be initially identified by itsassociation with an NTR protein. Wherein the BTRXh and NTR proteins andnucleic acids are novel, compositions and methods of use are providedherein. In the case that the BTRXh and NTR proteins and nucleic acidswere known but not known to be thioredoxins or oxidoreductases thatpreferably reduce thioredoxin h, methods of use, i.e. functionalscreens, are provided. In one embodiment, a BTRXh or an NTR protein asdefined herein has at least one of the following “BTRXh biologicalactivities or “NTR biologic activities”:

By “NTR biological activity” herein preferably is meant the catalyticreduction of thioredoxin coupled to NADPH₂ oxidation.

By “thioredoxin protein biological activity” herein is meant the abilityof a protein to serve as a hydrogen donor in various reduction reactions(Smith et al. (eds.) 1997). One of ordinary skill in the art will beaware that there are many well-established systems that can be employedto measure thioredoxin mediated reduction reactions. Preferred methodsof measuring biological thioredoxin activity attributable to thioredoxinh include NADP/malate dehydrogenase activation (Johnson et al., 1987)and reduction of 2′,5′-dithiobis(2-nitrobenzoic acid) (DTNB) viaNADP-thioredoxin reductase (Florencio et al., 1988; U.S. Pat. No.5,792,506). Due to the potential for interference from non-thioredoxin henzymes that use NADPH, accurate determination of thioredoxin h activityshould be made using partially purified plant extracts. Standard proteinpurification methods (e.g (NH₄)₂SO₄ extraction and acid fractionation)can be used to accomplish this partial purification, as discussed morefully below.

In one embodiment provided herein, BTRXh and an NTR protein as definedherein have sequence homology to other thioredoxin and NTR proteins,respectively. By “homology” herein is meant sequence similarity andidentity, with identity being preferred. In one embodiment, the homologyis found using the following database, algorithm and parameters.

The similarity between two nucleic acid sequences, or two amino acidsequences is expressed in terms of sequence identity (or, for proteins,also in terms of sequence similarity). Sequence identity is frequentlymeasured in terms of percentage identity; the higher the percentage, themore similar the two sequences are. As described above, homologs andvariants of the thioredoxin nucleic acid molecules, hordein promotersand hordein signal peptides may be used in the present invention.Homologs and variants of these nucleic acid molecules will possess arelatively high degree of sequence identity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman (1981); Needleman and Wunsch (1970); Pearson and Lipman(1988); Higgins and Sharp (1988); Higgins and Sharp (1989); Corpet etal., (1988); Huang et al., (1992); and Pearson et al., (1994). Altschulet al., (1994) presents a detailed consideration of sequence alignmentmethods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al.,1990) is available from several sources including the National Centerfor Biotechnology Information (NCBI, Bethesda, Md.) and on the internet,for use in connection with the sequence analysis programs blastp,blastn, blastx, tblastn and tblastx. It can be accessed atwww.ncbi.nlm.nih.gov/BLAST. A description of how to determine sequenceidentity using this program is available atwww.ncbi.nlm.nih.gov/BLAST/blast.help.html.

Homologs of the disclosed protein sequences are typically characterizedby possession of at least 40% sequence identity counted over the fulllength alignment with the amino acid sequence of the disclosed sequenceusing the NCBI Blast 2.0, gapped blastp set to default parameters. Theadjustable parameters are preferably set with the following values:overlap span=1, overlap fraction=0.125, word threshold (T)=11. The HSP Sand HSP S2 parameters are dynamic values and are established by theprogram itself depending upon the composition of the particular sequenceand composition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity. Proteins with even greater similarity to thereference sequences will show increasing percentage identities whenassessed by this method, such as at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 90% or at least about 95% sequence identity.

Homologs of the disclosed nucleic acid sequences are typicallycharacterized by possession of at least 40% sequence identity countedover the full length alignment with the amino acid sequence of thedisclosed sequence using the NCBI Blast 2.0, gapped blastn set todefault parameters. A preferred method utilizes the BLASTN module ofWU-BLAST-2 (Altschul et al., 1996); set to the default parameters, withoverlap span and overlap fraction set to 1 and 0.125, respectively.Nucleic acid sequences with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 90% orat least about 95% sequence identity.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the protein encoded by the sequences in thefigures, it is understood that in one embodiment, the percentage ofsequence identity will be determined based on the number of identicalamino acids in relation to the total number of amino acids. Thus, forexample, sequence identity of sequences shorter than that shown in thefigures as discussed below, will be determined using the number of aminoacids in the longer sequence, in one embodiment. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as, insertions, deletions, substitutions,etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0”,which obviates the need for a weighted scale or parameters as describedherein for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion. This method of sequence identity can be applied in the analysisof amino acid and nucleic acid sequences.

As will be appreciated by those skilled in the art, the sequences of thepresent invention may contain sequencing errors. That is, there may beincorrect nucleosides, frameshifts, unknown nucleosides, or other typesof sequencing errors in any of the sequences; however, the correctsequences will fall within the homology and stringency definitionsherein.

The alignment tools ALIGN (Myers and Miller, 1989) or LFASTA (Pearsonand Lipman, 1988) may be used to perform sequence comparisons (InternetProgram © 1996, W. R. Pearson and the University of Virginia,“fasta20u63” version 2.0u63, release date December 1996). ALIGN comparesentire sequences against one another, while LFAST compares regions oflocal similarity. These alignment tools and their respective tutorialsare available on the internet at biology.ncsa.uiuc.edu.

In a preferred embodiment, orthologs of the disclosed barley thioredoxinh protein are typically characterized by possession of greater than90.6% sequence identity counted over the full-length alignment with theamino acid sequence of barley thioredoxin h using ALIGN set to defaultparameters. Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 92%, at least 93%, at least 95%, at least96%, at least 97%, at least 98% sequence identity. When less than theentire sequence is being compared for sequence identity, homologs willtypically possess at least 90% sequence identity over short window of10-20 amino acids, and may possess sequence identities of at least 93%,at least 95%, at least 97%, or at least 99% depending on theirsimilarity to be reference sequence. Sequence identity over such shortwindows can be determined using LFASTA; methods are described atbiology.ncsa.uiuc.edu. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided. The present invention providesnot only the peptide homologs that are described above, but also nucleicacid molecules that encode such homologs.

In a preferred embodiment, members of a thioredoxin h protein familyhaving thioredoxin protein biological activity sharing amino acidsequence identity with the amino acid sequence of the prototypicalbarley thioredoxin h protein shown in SEQ ID NO:2. In a preferredembodiment, BTRXh proteins of the invention will generally share greaterthan 90.2% amino acid sequence identity with the sequence shown in SEQID NO:2, as determined using ALIGN set to default parameters. Moreclosely related thioredoxin proteins may share at least 92%, 95%, or 98%sequence identity with the exemplified BTRXh protein.

In a preferred embodiment, a protein is a “NTR protein” as definedherein if the overall sequence identity of the amino add sequence ofFIG. 4A (SEQ ID NO:9) is preferably greater than about 71%, morepreferably greater than about 85%, even more preferably greater thanabout 90% and most preferably greater than 95%. In some embodiments thesequence identity will be as high as about 98% and higher.

Barley thioredoxin h derived proteins and NTR derived proteins includefragments, respectively of thioredoxin h or NTR, generated either bychemical (e.g. enzymatic) digestion or genetic engineering means;chemically functionalized protein molecules obtained starting withdisclosed protein or nucleic acid sequences, and protein sequencevariants, which retain measurable thioredoxin h protein biologicalactivity.

For example, while the prototypical barley thioredoxin h protein shownin SEQ ID NO:2 is 122 amino acids in length, one of skill in the artwill appreciate that thioredoxin biological activity may be obtainedusing a protein that comprises less than the full length barleythioredoxin h protein. Thus the terms “barley thioredoxin h protein” and“barley NTR protein” includes fragments, respectively, of a full lengthbarley thioredoxin h protein, which fragments retain thioredoxin proteinbiological activity or NTR protein biological activity, and variants,such as, naturally occurring allelic variants and mutants obtained by invitro mutagenesis techniques and the like as further described below.

In one embodiment, BTRXh and NTR nucleic acids or BTRXh and NTR proteinsare initially identified by substantial nucleic acid and/or amino acidsequence identity or similarity to the sequence(s) provided herein. In apreferred embodiment, BTRXh or NTR nucleic acids or Brtxh or NTRproteins have sequence identity or similarity to the sequences providedherein and one or more of their respective “biological activities”. Suchsequence identity or similarity can be based upon the overall nucleicacid or amino acid sequence.

The BTRXh and NTR proteins of the present invention may be shorter orlonger than the amino acid sequence encoded by the exemplified nucleicacids shown in SEQ ID NO:2 and SEQ ID NO:9. Thus, in a preferredembodiment, included within the definition of BTRXh or NTR proteins areportions or fragments of the respective amino acid sequence encoded bythe nucleic acid sequence provided herein. In one embodiment herein,fragments of BTRXh or NTR proteins are considered BTRXh or NTR proteinsif a) a fragment shares at least one antigenic epitope with thecorresponding exemplified sequence; b) has at least the indicatedsequence homology; and c) preferably have an BTRXh or NTR biologicalactivity or enzymatic activity as further defined herein. In some cases,where the sequence is used diagnostically, that is, when the presence orabsence of a BTRXh or an NTR protein nucleic acid is determined, onlythe indicated sequence identity is required. The nucleic acids of thepresent invention may also be shorter or longer than the exemplifiedsequences in FIG. 2 (SEQ ID NO:1) or FIG. 5A (SEQ ID NO:10). The nucleicacid fragments include any portion of the nucleic acids provided hereinwhich have a sequence not exactly previously identified; fragmentshaving sequences with the indicated sequence identity to that portionnot previously identified are provided in an embodiment herein.

In addition, as is more fully outlined below, a BTRXh or an NTR proteincan be made that are longer than those depicted in FIG. 1 (SEQ ID NO:2)and FIG. 4 (SEQ ID NO:9); for example, by the addition of epitope orpurification tags, the addition of other fusion sequences, or theelucidation of additional coding and non-coding sequences. As describedbelow, the fusion of a NTR peptide to a fluorescent peptide, such asGreen Fluorescent Peptide (GFP), is particularly preferred.

BTRXh or NTR proteins may also be identified as encoded by BTRXh or NTRnucleic acids which hybridize to the sequence depicted in the FIG. 2(SEQ ID NO:1) or FIG. 5A (SEQ ID NO:10) or the complement thereof, asoutlined herein. Hybridization conditions are further described below.

In a preferred embodiment, when a BTRXh or NTR protein is to be used togenerate antibodies, a BTRXh or an NTR protein must share at least oneepitope or determinant with the full length protein. By “epitope” or“determinant” herein is meant a portion of a protein which will generateand/or bind an antibody. Thus, in most instances, antibodies made to asmaller BTRXh or NTR protein will be able to bind to the full lengthprotein. In a preferred embodiment, the epitope is unique; that is,antibodies generated to a unique epitope show little or nocross-reactivity. The term “antibody” includes antibody fragments, asare known in the art, including Fab, Fab₂, single chain antibodies (Fvfor example), chimeric antibodies, etc., either produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA technologies (Harlow & Lane, 1988).

In a preferred embodiment, an antibody to an BTRXh or NTR protein uponbinding to an NTR protein reduce or eliminate at least one biologicalactivity of the NTR protein as described herein. That is, the additionof anti BTRXh or an anti-NTR protein antibodies (either polyclonal orpreferably monoclonal) to anti-Btrxh or NTR proteins (or cellscontaining Brtxh or NTR proteins) may reduce or eliminate a BTRXh or anNTR activity. Generally, for both proteins of the invention at least a25% decrease in activity is preferred, with at least about 50% beingparticularly preferred and about a 95-100% decrease being especiallyreferred.

The antibodies of the invention specifically bind to either BTRXh or NTRproteins. By “specifically bind” herein is meant that an antibody bindto a protein with a binding constant in the range of at least 10⁻⁴-10⁻⁶M⁻¹, with a preferred range being 10⁻⁷-10⁻⁹ M⁻¹. Antibodies are furtherdescribed below.

In the case of the BTRXh or NTR nucleic acid, the overall sequenceidentity of the nucleic acid sequence is commensurate with amino acidsequence identity but takes into account the degeneracy in the geneticcode and codon bias of different organisms. Accordingly, the nucleicacid sequence identity may be either lower or higher than that of theenocoded protein sequence.

Thus the NTR nucleic acid sequence identity of the nucleic acid sequenceas compared to the nucleic acid sequence of the Figures is preferablygreater than 75%, more preferably greater than about 80%, particularlygreater than about 85% and most preferably greater than 90%. In someembodiments the sequence identity will be as high as about 93 to 95 or98%.

In a preferred embodiment, a NTR nucleic acid encodes a NTR protein;whereas a BTRXh nucleic acid encodes a BTRXh protein. As will beappreciated by those in the art, due to the degeneracy of the geneticcode, an extremely large number of nucleic acids may be made, all ofwhich encode either the BTRXh or the NTR proteins of the presentinvention. Thus, having identified a particular amino acid sequence,those skilled in the art could make any number of different nucleicacids, by simply modifying the sequence of one or more codons in a waywhich does not change the amino acid sequence of the encoded protein.

In one embodiment, the BTRXh or the NTR nucleic acid is determinedthrough hybridization studies. Thus, for example, nucleic acids whichhybridize under high stringency to the nucleic acid sequence shown inFIG. 2 (SEQ ID NO:1) or FIG. 5A (SEQ ID NO:10), or their complement areconsidered either a BTRXh or an NTR nucleic acid. High stringencyconditions are known in the art; see for example Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and ShortProtocols in Molecular Biology, ed. Ausubel, et al., and Tijssen,Techniques in Biochemistry and Molecular Biology-Hybridization withNucleic Acid Probes, “Overview of principles of hybridization and thestrategy of nucleic acid assays” (1993), all of which are herebyincorporated by reference in their entirety. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences specifically hybridize at higher temperatures.Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic acid concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Thus, it is known in theart that hybridization stringency is an objective measure of sequencerelatedness. Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at about pH 7.0 to8.3 and the temperature is at least about 30° C. for short probes (e.g.about 10 to 50 nucleotides) and at least about 60° C. for long probes(e.g. greater than about 50 nucleotides). Stringent conditions may alsobe achieved with the addition of destabilizing agents such as formamide.In a preferred embodiment, high stringency conditions are 0.1×SSC at 65°C.

In another embodiment, less stringent hybridization conditions are used;for example, moderate or low stringency conditions may be used, as areknown in the art; see Maniatis and Ausubel, supra, and Tijssen, supra.

In a preferred embodiment, the BTRXh and NTR proteins and nucleic acidsof the present invention are recombinant. As used herein and furtherdefined below, “nucleic acid” may refer to either DNA or RNA, ormolecules which contain both deoxy- and ribonucleotides. The nucleicacids include genomic DNA, cDNA and oligonucleotides including sense andanti-sense nucleic acids. Such nucleic acids may also containmodifications in the ribose-phosphate backbone to increase stability andhalf life of such molecules in physiological environments.

The nucleic acid may be double stranded, single stranded, or containportions of both double stranded or single stranded sequence. As will beappreciated by those in the art, the depiction of a single strand(“Watson”) also defines the sequence of the other strand (“Crick”); thusthe sequences depicted in the Figures also include the complement of thesequence. By the term “recombinant nucleic acid” herein is meant nucleicacid, originally formed in vitro, in general, by the manipulation ofnucleic acid by an endonuclease and/or a polymerase and/or a ligaseand/or a recombinase, in a form not normally found in nature. Thus arecombinant BTRXh or NTR nucleic acid, in a linear form, or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined, are both considered recombinant for the purposes ofthis invention. It is understood that once a recombinant nucleic acid ismade and reintroduced into a host cell or organism, it will replicatenon-recombinantly, i.e. using the in vivo cellular machinery of the hostcell rather than in vitro manipulations; however, such nucleic acids,once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposes ofthe invention.

Similarly, a “recombinant protein” is a protein made using recombinanttechniques, i.e. through the expression of a recombinant nucleic acid asdescribed herein. A recombinant protein is distinguished from naturallyoccurring protein by at least one or more characteristics for example,the protein may be isolated or purified away from some or all of theproteins and compounds with which it is normally associated in its wildtype host, and thus may be substantially pure. For example, an isolatedprotein is unaccompanied by at least some of the material with which ofis normally associated in its natural state, preferably constituting atleast about 0.5%, more preferably at least about 5% by weight of thetotal protein in a given sample. A substantially pure protein comprisesat least about 75% by weight of the total protein, with at least about80% being preferred, and at least about 90% being particularlypreferred. The definition includes the production of a BTRXh or NTRprotein from one organism in a different organism or host cell.Alternatively, the protein may be made at a significantly higherconcentration than is normally seen, through the use of an induciblepromoter or high expression promoter or by increasing the number ofcopies of a nucleic acid encoding the BTRXh or NTR protein, such thatthe protein is made at increased concentration levels. Alternatively,the protein may be in a form not normally found in nature, as in theaddition of an epitope tag or amino acid substitutions, insertions,and/or deletions, as discussed below.

In one embodiment, the present invention provides BTRXh and NTR proteinvariants. These variants fall into one or more of three classes:substitutional, insertional or deletional variants. These variantsordinarily are prepared by site specific mutagenesis of nucleotides inthe DNA encoding a BTRXh or an NTR protein, using cassette mutagenesis,alanine scanning mutagenesis, glycine scanning mutagenesis, PCRmutagenesis, gene shuffling or other techniques well known in the art,to produce a nucleic acid encoding the variant, and thereafterexpressing the nucleic acid in recombinant host cell culture as outlinedabove. However, variant BTRXh or NTR protein fragments having up toabout 100-150 residues may be prepared by in vitro synthesis usingestablished techniques. Amino acid sequence variants are characterizedby the predetermined nature of the variation, a feature that sets themapart from naturally occurring allelic or interspecies variation of theBTRXh or NTR protein amino acid sequence. The variants typically exhibitthe same qualitative biological activity as the naturally occurringanalogue, although variants can also be selected which have modifiedcharacteristics as will be more fully outlined below.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed BTRXh or NTR variants screenedfor the optimal combination of desired activity.

Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known and include, for example, M13primer mutagenesis, PCR mutagenesis, gene shuffling. Screening of themutants is done using assays of BTRXh or NTR protein activities and/orproperties as defined herein.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arive at a final derivative. Generally these changes are done ona few amino acids to minimize the alteration of the molecule. However,larger changes may be tolerated in certain circumstances. When smallalterations in the characteristics of the BTRXh or NTR protein aredesired, substitutions are generally made in accordance with thefollowing chart:

CHART I Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln,His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart I. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g. seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,phenyfalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine.

Covalent modifications of BTRXh or NTR polypeptides are included withinthe scope of this invention. One type of covalent modification includesreacting targeted amino acid residues of a BTRXh or NTR polypeptide withan organic derivatizing agent that is capable of reacting with selectedside chains or the N-or C-terminal residues of a BTRXh or NTRpolypeptide. Derivatization with bifunctional agents is useful, forinstance, for crosslinking a BTRXh or NTR protein to a water-insolublesupport matrix or surface for use in the method of purifying anti-Btrxhor anti-NTR antibodies or screening assays, as is more fully describedbelow. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidyl-propionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of the “aminogroups of lysine, arginine, and histidine side chains [T. E. Creighton,Proteins: Stucture and Molecular Properties, W. H. Freeman & Co., SanFrancisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, andamidation of any C-terminal carboxyl group.

Another type of covalent modification of the BTRXh or NTR polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence of a BTRXh orNTR polypeptide, if present, and/or adding one or more glycosylationsites that are not present in the native sequence BTRXh or NTRpolypeptide.

Addition of glycosylation sites to Brtxh or NTR polypeptides may beaccomplished by altering the amino acid sequence thereof. The alterationmay be made, for example, by the addition of, or substitution by, one ormore serine or threonine residues to the native sequence BTRXh or NTRpolypeptide (for O-linked glycosylation sites). The alteration also maybe made, for example, by the addition of, or substitution by one or moreAxn-Xaa-Ser/Thr sites (Xaa=any amino acid) in the native sequence BTRXhor NTR polypeptide (for N-linked glycosylation sites). The STRXh or NTRamino acid sequence may optionally be altered through changes at the DNAlevel, particularly by mutating the DNA encoding the BTRXh or NTRpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theBTRXh or NTR polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Such methods are described in the art,e.g., in WO 87/05330 published 11 September 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the BTRXh or NTR polypeptidemay be accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., ArchBiochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties if presenton polypeptides or variant polypeptides can be achieved by the use of avariety of endo-and exo-glycosidases as described by Thotakura et al.,Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of BTRXh or NTR polypeptidecomprises linking the BTRXh or NTR polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, or polyoxyalkylines, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

BTRXh or NTR polypeptides of the present invention may also be modifiedin a way to form chimeric molecules comprising an BTRXh or NTRpolypeptide fused to another, heterologous polypeptide or amino acidsequence. Encompassed within this embodiment are Btrxh-NTR fusions. Inone embodiment, such a chimeric molecule comprises a fusion of a BTRXhor NTR polypeptide with a tag polypeptide which provides an epitope towhich an anti-tag antibody can selectively bind. The epitope tag isgenerally placed at the amino-or carboxyl-terminus of the BTRXh or NTRpolypeptide but may be incorporated as an internal insertion orsubstitution. The presence of such epitope-tagged forms of a BTRXh orNTR polypeptide can be detected using an antibody against the tagpolypeptide. Also, provision of the epitope tag enables the BTRXh or NTRpolypeptide to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. In an alternative embodiment, the chimeric molecule maycomprise a fusion of a BTRXh or NTR polypeptide with an immunoglobulinor a particular region of an immunoglobulin. For a bivalent form of thechimeric molecule, such a fusion could be to the Fc region of an IgGmolecule as discussed further below.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Bio., 8:2159-2165(1988); the c-myc tag and the 8F9, 3C7. 6E10, G4, B7 and 9E10 antibodiesthereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616(1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and itsantibody [Paborsky et al., Protein Engineering. 3(6):547-553 (1990)].Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al.,Science. 255:192-194 (1992)]; tubulin epitope peptide [Skinner et al.,J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 proteinpeptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)] and the histidine tag and metal binding sites(Smith, Ann. NY. Acad. Sci., 646:315-321 (1991)], with the Flag andhistidine tag being preferred.

In an embodiment herein, nucleic acids comprising sequences homologousto the exemplified BTRXh and NTR proteins of other organisms or tissuesor alleles are cloned and expressed as outlined below. Thus, probe ordegenerate polymerase chain reaction (PCR) primer sequences may be usedto find other related BTRXh or NTR proteins from humans or otherorganisms. As will be appreciated by those in the art, particularlyuseful probe and/or PCR primer sequences include the unique areas of theBTRXh or NTR nucleic acid sequence. As is generally known in the art,preferred PCR primers are from about 15 to about 35 nucleotides inlength, with from about 20 to about 30 being preferred, and may containinosine as needed. The conditions for the PCR reaction are well known inthe art (Innis et al., 1990). It is therefore also understood thatprovided along with the sequences in the sequences listed herein areportions of those sequences, wherein unique potions of 15 nucleotides ormore are particular preferred. The skilled artisan can routinelysynthesize or cut a nucleotide sequence to the desired length.

Once isolated from its natural source, e.g., contained within a plasmidor other vector or excised therefrom as a linear nucleic acid segmentthe recombinant Brtxh or NTR nucleic acid can be further-used as a probeto identify and isolate related Brtxh or NTR nucleic acids. It can alsobe used as a “precursor” nucleic acid to make modified or variant BTRXhor NTR nucleic acids and proteins.

Using the nucleic acids of the present invention which encode an BTRXhor NTR protein, a variety of expression vectors are made. The expressionvectors may be either self-replicating extrachromosomal vectors orvectors which integrate into a host genone. Generally, these expressionvectors include transcriptional and translational regulatory nucleicacid operably linked to the nucleic acid encoding the BTRXh or NTRprotein. The term “control sequenes” refers to nucleic acid sequencesnecessary for the expression of an operably linked coding sequence in aparticular host organism. The control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, and a ribosome binding site. Eukaryotic cells are known toutilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. As another example, operablylinked refers to DNA sequences linked so as to be contiguous, and, inthe case of a secretory leader, contiguous and in reading phase.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice. The transcriptional and translationalregulatory nucleic acid will generally be appropriate to the host cellused to express the BTRXh or NTR protein; for example, transcriptionaland translational regulatory nucleic acid sequences from barley arepreferably used to express the NTR protein in barley. Numerous types ofappropriate expression vectors, and suitable regulatory sequences areknown in the art for a variety of host cells.

In general, the transcriptional and translational regulatory sequencesmay include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences. In apreferred embodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters.The promoters may be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known in the art, and are useful in the presentinvention.

In addition, the expression vector may comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in mammalianor plant cells for expression and in a procaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequenes which flank theexpression construct. The integrating vector may be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

In addition, in a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

A preferred expression vector system for use in plant cells and forproduction of transgenic plants are provided herein and in the Examples.

BTRXh or NTR proteins of the present invention are produced by culturinga host cell transformed with an expression vector containing nucleicacid encoding a BTRXh or NTR protein, under the appropriate conditionsto induce or cause expression of the BTRXh or NTR protein. Theconditions appropriate for BTRXh or NTR protein expression will varywith the choice of the expression vector and the host cell, and will beeasily ascertained by one skilled in the art through routineexperimentation. For example, the use of constitutive promoters in theexpression vector will require optimizing the growth and proliferationof the host cell, while the use of an inducible promoter requires theappropriate growth conditions for induction. In addition, in someembodiments, the timing of the harvest is important. For example, thebaculoviral systems used in insect cell expression are lytic viruses,and thus harvest time selection can be crucial for product yield.

Appropriate host cells include plant, yeast, bacteria, archebacteria,fungi, insect and animal cell, including mammalian cells. Of particularinterest are plant embryos, plant seeds and grains, root cells, stemcells, leaf cells, and other plant cells, Drosophila melangaster cells,Saccharomyces cerevisiae and other yeast, E. coli, Bacillus subtilis,SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLacells, fibroblasts, Schwanoma cell lines, immortalized mammalian myeloidand lymphoid cell lines, with HeLa, SF9, and plant cells beingpreferred.

In a preferred embodiment, the BTRXh or NTR proteins are expressed inseed, grain, root, stem, leaf cells etc of dicotyledonous plants andmonocotyledonous plants. Thus, BTRXh and NTR are expressed, for example,in wheat (Triticum spp.), rice (Oryza spp.), barley (Hordeum spp.), oat(Avena spp.), rye (Secale spp.), maize, corn (Zea mays), sorghum(Sorghum spp.), millet (Pennisetum spp.), Brassica spp., soybean,cotton, beans in general, rape/canola, alfalfa flax, sunflower,safflower, cotton, tobacco, flax, peanut, clover, cowpea, grapes,forages grass varieties; vegetables such as lettuce, tomato, curcurbits,cassava, potato, carrot, radish, pea, lentils, cabbage, sugar beets,cauliflower, broccoli, sugar beats, Brussels sprouts, peppers; treefruits such as citrus, apples, pears, peaches, apricot, walnuts; andornamentals such as turf grasses, carnations and roses. In a preferredembodiment, the present invention can be employed with barley genotypesincluding, but not limited to Morex, Harrington, Crystal, Stander,Moravian III, Galena, Salome, Steptoe, Klages, Baronesse, and with wheatgenotypes including, but not limited to Yecora Rojo, Bobwhite, Karl andAnza. In general, the invention is particularly useful in cereals.

A number of recombinant vectors suitable for stable transfection ortransformation of plant cells or for the establishment of transgenicplants have been described including those described in Weissbach andWeissbach (1989), and Gelvin et al. (1990). Typically, planttransformation vectors include one or more cloned plant genes (or cDNAs)under the transcriptional control of 5′ and 3′ regulatory sequences, anda dominant selectable marker. Such plant transformation vectorstypically also contain a promoter regulatory region (e.g., a regulatoryregion controlling inducible or constitutive, environmentally ordevelopmentally regulated, or cell- or tissue-specific expression), atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and/or apolyadenylation signal.

Examples of constitutive plant promoters that may be useful forexpressing the an operatively linked nucleic acid include: thecauliflower mosaic virus (CaMV) 35S promoter, which confersconstitutive, high-level expression in most plant tissues (see, e.g,Odel et al., 1985, Dekeyser et al., 1990, Terada and Shimamoto, 1990,Benfey and Chua, 1990); the nopaline synthase promoter (An et al.,1988); the maize ubiquitin promoter (Christianson & Quail, 1996) and theoctopine synthase promoter (Fromm et al., 1989).

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals, alsocan be used for expression of the Btrxh or NTR nucleic acid in plantcells, including promoters regulated by: (a) heat (Callis et al. 1988;Ainley, et al. 1993; Gilmartin et al. 1992); (b) light (e.g., the pearbcS-3A promoter, Kuhlemeier et al., 1989, and the maize rbcS promoter,Schaffner and Sheen, 1991); (c) wounding (e.g., wunl, Siebertz et al., 19893; (d) hormones, such as abscisic acid (Marcotte et al., 1989); and(a) chemicals such as methyl jasminate or salicylic acid (see also Gatz,1997).

In an alternative embodiment, tissue or organ specific (root, leaf,flower, and seed for example) promoters (Carpenter et al., 1992; Deniset al., 1993; Opperman et al., 1994; Stockhauser et al., 1997; Roshal etal., 1987; Schernthaner et al., 1988; Bustos et al., 1989) can beoperably linked to the coding sequence to obtain particular expressionin respective organs. For instance, monocot tissue-specific promotersmay be used to attain expression in the aleurone (U.S. Pat. No.5,525,716) or the endosperm (U.S. Pat. No. 5,677,474) of cereal andother grains.

In a preferred embodiment, a transgene of the invention, the Btrwh orNTR nucleic acid, is expressed in an edible part of a plant. By “edible”herein is meant at least a part of a plant that is suitable forconsumption by humans or animals (fish, crustaceans, isopods, decapods,monkeys, cows, goats, pigs, rabbits, horses, birds (chickens, parrotsetc). Accordingly, “edible” embraces food for human consumption and feedfor animal consumption and includes, for example, dough, bread, cookies,pasta, pastry, beverages, beer, food additives, thickeners, malt,extracts made from an edible part of plants, animals feeds, and thelike. An edible part of a plant includes for example, a root, a tuber, aseed, grain, a flower, fruit, leaf etc. The skilled artisan is awarethat expression of the transgene is effected in any tissue, organ orpart of a plant by employing a promoter that is active in the selectedpart of the plant the transgene is to be expressed. In a preferredembodiment the transgene is expressed in a seed or grain, preferablyunder control of a seed- or grain-specific promoter.

The expression of a Btrwh or NTR nucleic acid transgene in seeds orgrains according to the present invention is preferably accomplished byoperably linking a seed-specific or grain-specific promoter to thenuclei acid molecule encoding the transgene Btrwh or NTR protein. Inthis context, “seed-specific” indicates that the promoter has enhancedactivity in seeds compared to other plant tissues; it does not requirethat the promoter is solely active in the seeds. Accordingly,“grain-specific” indicates that the promoter has enhanced activity ingrains compared to other plant tissues; it does not require that thepromoter is solely active in the grain. Preferably, the seed- orgrain-specific promoter selected will, at the time when the promoter ismost active in seeds, produce expression of a protein in the seed of aplant that is at least about two-fold greater than expression of theprotein produced by that same promoter in the leaves or roots of theplant, However, given the nature of the Btrwx and NTR protein, it may beadvantageous to select a seed- or grain-specific promoter that causeslittle or no protein expression in tissues other than seed or grain. Ina preferred embodiment, a promoter is specific for seed and grainexpression, such that, expression in the seed and grain is enhanced ascompared to other plant tissues but does not require that the promoterbe sole activity in the grain or seed. In a preferred embodiment, thepromoter is “specific” for a structure or element of a seed or grain,such as an embryo-specific promoter. In accordance with the definitionsprovided above, an embryo-specific promoter has enhanced activity in anembryo as compared to other parts of a seed or grain or a plant and doesnot require its activity to be limited to an embryo. In a preferredembodiment, the promoter is “maturation-specific” and accordingly hasenhanced activity developmentally during the maturation of a part of aplant as compared to other parts of a plant and does not require itsactivity to be limited to the development of one part of a plant.

A seed- or grain-specific promoter may produce expression in variousparts of the seed or grain, including the endosperm, embryo, aleuroneetc. or grain. Any seed- or grain-specific promoter may be used for thispurpose, although it will be advantageous to select a seed- orgrain-specific promoter that produces high level expression of theprotein in the plant seed or grain. Known seed- or grain-specificpromoters include those associated with genes that encode plant seedstorage proteins such as genes encoding; barley hordeins, riceglutelins, oryzins, or prolamines; wheat gliadins or glutenins; maizezeins or glutelins; maize embryo-specific promoter; oat glutelins;sorghum kafirins; millet pennisetins; or rye secalins.

The barley hordein promoters (described in more detail below) are seed-or grain-specific promoters that were used in the illustrative Examples.

In certain embodiments, the seed- or grain-specific promoter that isselected is a maturation-specific promoter. The use of promoters thatconfer enhanced expression during seed or grain maturation (such as thebarley hordein promoters) may result in even higher levels ofthioredoxin expression in the seed.

By “seed or grain-maturation” herein refers to the period starting withfertilization in which metabolizable food reserves (e.g., proteins,lipids, starch, etc.) are deposited in the developing seed, particularlyin storage organs of the seed, including the endosperm, testa, aleuronelayer, embryo, and scutellar epithelium, resulting in enlargement andfilling of the seed and ending with seed desiccation.

Members of the grass family, which include the cereal grains, producedry, one-seeded fruits. This type of fruit, is strictly speaking, acaryopsis but is commonly called a kernel or grain. The caryopsis of afruit coat or pericarp, which surrounds the seed and adhere tightly to aseed coat. The seed consist of an embryo or germ and an endospermenclosed by a nucellar epidermis and a seed coat. Accordingly the graincomprises the seed and its coat or pericarp. The seed comprise theembryo and the endosperm (R. Carl Hoseney in “Principles of CerealScience and Technology”, expressly incorporated by reference in itsentirety).

In a preferred embodiment a hordein promoter is operably linked to aBTRXh or NTR nucleic acid. By “hordein promoter” and grammaticalequivalents herein is meant, a barley promoter that directstranscription of a hordein gene in barley seeds or grain. A number ofbarley hordein genes and associated promoters have been described andcharacterized, including those for the B-, C-, D-, and Gamma-hordeins(Brandt et al., 1985, Forde et al., 1985, ; Rasmussen and Brandt, 1986,Sørensen et al., 1996). The activities of these promoters in transientexpression assays have also been characterized (Entwistle et al., 1991,Muller and Knudesen, 1993; Sørensen et al., 1996). While any hordeinpromoter may be employed for this invention, the specific Examplesprovided describe the use of the sequences from the B₁- and D-hordeingenes of barley. The nucleic acid sequences of the barley B₁- andD-hordein genes are shown in SEQ ID NOs:11 and 12, respectively and inFIGS. 11 and 12 (the promoter region excludes those nucleotide thatencode the hordein signal peptide that is shown underlined). Sørensen etal., (1996) describes plasmids that comprise the B₁- and D-hordeinpromoters operably linked to a beta-glucuronidase gene (uidA; gus) andthe Agrobacterium tumefaciens nopaline synthase 3′polyadenylation site(nos). These plasmids may be conveniently utilized as sources of boththe hordein promoters and the nos polyadenylation site.

One of skill in the art will appreciate that the length of the hordeinpromoter region may also be greater or less than the sequence depictedin FIGS. 11 and 12. For example, additional 5′sequence from the hordeingene upstream region may be added to the promoter sequence, or bases maybe removed from the depicted sequences. However, any hordein promotersequence must be able to direct transcription of an operably linkedsequence in plant seed or grain. The ability of a barley hordeinpromoter to direct transcription of a protein in a plant seed mayreadily be assessed by operably linking the promoter sequence to an openreading frame (ORF) that encodes a readily detctable protein, such asthe gus ORF, introducing the resulting construct into plants and thenassessing expression of the protein in seeds of the plant (see Sørensenet al., 1998). A hordein promoter will typically confer seed-specificexpression, meaning that expression of the protein encoded by theoperably linked ORF will generally be at least about twice as high(assessed on an activity basis) in seeds of the stably transfected plantcompared to other tissues such as leaves. More usually, the hordeinpromoter will produce expression in seeds that is at least about 5 timeshigher than expression in other tissues of the plant.

Functional homologs of the barley hordein promoters disclosed herein maybe obtained from other plant species, such as from other monocots,including wheat, rice and corn. Such homologs may have specified levelsof sequence identity with the prototype hordein promoters (e.g., atleast 40% sequence identity). The functional homologs retain hordeinpromoter function, i.e., retain the ability to confer seed- orgrain-specific expression of operably linked ORFs when introduced intoplants (Marris et al., 1988; Mena et al., 1998). Accordingly, wherereference is made herein to a hordein promoter, it will be understoodthat such reference includes not only nucleic acid molecules having thesequences of the prototypical sequences disclosed herein (or variationson these sequences), but also promoters from hordein gene homologs. Alsoincluded within the scope of such terms are molecules that differ fromthe disclosed prototypical molecules by minor variations. Such variantsequences may be produced by manipulating the nucleotide sequence ofhordein promoter using standard procedures such as site-directedmutagenesis or the polymerase chain reaction. Preferably, the seed- orgrain-specificity of the promoter is retained. Examples of dicotpromoters that can be used include for example soybean glycinins andcon-glycinins, and kidney bean phaseolin promoters.

In a preferred embodiment, the vector for plant expression of BTRXh andNTR polypeptides comprises a signal sequence which encodes a signalpeptide. As described in the Examples below, the inventors havediscovered that the level of expression of a transgene in seed or graincan be enhanced by the presence of a signal peptide. In one of theExamples described below, the B₁ hordein signal peptide was utilized. Inparticular, it was discovered that the expression of thioredoxin proteinin seed or grain is enhanced when the ORF encoding the protein isoperably linked to both a hordein promoter and a hordein signal sequenceencoding the signal peptide. (For convenience, the nucleic acid sequenceencoding a signal peptide is referred to herein as a signal sequence(SS).) While not wishing to be bound by theory, it is proposed that thehordein signal peptide directs expression of the thioredoxin protein toa protected subcellular location, such as a vacuole or protein body. Itis further proposed that proteins directed to such vacuoles areprotected from proteolysis during certain stages of seed or grainmaturation. In addition, the sequestration of the BTRXh or NTR proteinto such a location may also serve to protect the maturing seeds or grainfrom detrimental effects associated with over-expression of saidproteins.

The hordein signal peptide typically comprises about the first 15-25amino acids of the hordein gene ORF, more usually about 18-21 aminoacids. The nucleotide and amino acid sequences of the hordein signalsequence and peptide of the prototypical barley B1- and D-hordein genesare shown in SEQ ID NO:11-12 and FIGS. 11 and 12. One of skill in theart will appreciate that while the B₁-hordein signal sequence and signalpeptide are utilized in the examples described below, the invention isnot limited to these specific sequences. For example, homologoussequences may be used as effectively, as may sequences that differ inexact nucleotide or amino acid sequences, provided that such sequencesresult in enhanced levels of the encoded protein in immature seed orgrain. Typically, “enhanced expression” will be expression that is abouttwice that observed with an equivalent construct lacking the signalsequence. Accordingly, the term “hordein signal sequence” and “horteinsignal peptide” includes not only the particular sequences shown herein,but also homologs and variants of these sequences.

Furthermore, the invention is not limited to the use of hordein signalpeptides. Other signal peptides that serve to localize the thioredoxinco-translationally or post-translationally to a selected seed, grain orcell compartment may be employed. Other such signal sequences includethose associated with storage proteins in maize, rice, wheat, soybeans,beans, and tobacco (see for example: Bagga et al., 1997; Torrent et al.,1997; Wu et al., 1998; Zheng et al., 1995; Grimwade et al., 1996; Conradet al., 1998; and Takaiwa et al., 1995.)

In a preferred embodiment, plant transformation vectors may also includeRNA processing signals, for example introns, which may be positionedupstream or downstream of the ORF sequence in the transgene. Inaddition, the expression vectors may also include additional regulatorysequences from the 3′ untranslated region of plant genes, e.g., a 3′terminator region to increase stability of the mRNA, such as the PI-IIterminator region of potato or the octopine or nopaline synthase (nos)3′ terminator regions.

Finally, as noted above, plant transformation vectors may also includedominant selectable marker genes to allow for the ready selection oftransformants. Such genes include those encoding antibiotic resistancegenes (e.g, resistance to hygromycin, kanamycin, bleomycin, G418,streptomycin or spectinomycin) and herbicide resistance genes (e.g.,phosphinothricin acetyltransferase).

The vector and transcriptional regulatory elements used for transgeneexpression is selected at the discretion of the practitioner. In someinstances, enhanced BTRXh or NTR polypeptide expression and/or activityis desired, and the respective transgene encoding sequence is operablylinked to a high-level promoter such as the maize ubiquitin 1 promoter.Enhanced BTRXh or NTR activity may also be achieved by introducing intoa plant a transformation vector containing a variant form of the BTRXhor NTR polypeptide encoding sequence, for example a form which variesfrom an exemplified sequence but encodes a protein that retains BTRXh orNTR biological activity

Over-expression of BTRXh or NTR in plant or other type of eukaryotic orprocaryotic expression system is usually measured as the increase in theBTRXh or NTR activity present in a sample. Such over-expression can bemeasured using standard thioredoxin act and NTR activity assays. As usedhere, cells, tissues, or plants over-expressing BTRXh or NTR, orhomologous or derived proteins having BTRXh or NTR polypeptide activity,generally will have activity levels attributable of at least 5% overthat found in the equivalent wild-type (nontransformed) sample. Whereparticularly high levels of over-expression are desired, transformedcells will express at least 30%, more preferably at least 50%, even morepreferred at least 70%, or most preferred at least 100% more thioredoxinor NTR activity attributable in comparison to an equivalent wild-type ornull segregant sample. Overexpression of BTRXh or NTR polypeptideactivity also may be measured by assessing the amount of protein inplant tissues using well-known procedures.

In an alternative embodiment, a reduction of BTRXh or NTR activity,preferably in a transgenic plant, may be obtained by introducing intoplants an antisense construct based on a BTRXh or NTR encoding sequence.For antisense suppression, a BTRXh or NTR encoding sequence is arrangedin reverse orientation relative to the promoter sequence in thetransformation expression vector. The introduced sequence need not be afull length barley thioredoxin h or NTR encoding sequence, and need notbe exactly homologous to the native thioredoxin h or NTR cDNA or genefound in the plant species, type, cultivar, varietal, or subspecies tobe transformed. Generally, however, where the introduced sequence is ofshorter length, a higher degree of homology to the native thioredoxinsequence will be needed for effective antisense suppression.

The introduced antisense sequence in the vector generally will be atleast 30 nucleotides in length, and improved antisense suppression willtypically be observed as the length of the antisense sequence increases.Preferably, the length of the antisense sequence in the vector will begreater than 100 nucleotides. Transcription of an antisense construct asdescribed results in the production of RNA molecules that are thereverse complement of mRNA molecules transcribed from the endogenousthioredoxin or NTR gene in the plant cell. Although the exact mechanismby which antisense RNA molecules interfere with gene expression has notbeen elucidated, without being bound by theory, the antisense RNAmolecules bind to the endogenous mRNA molecules and thereby inhibittranslation of the endogenous mRNA. The production and use of antisenseconstructs are disclosed, for instance, in U.S. Pat. Nos. 5,773,692(using constructs encoding anti-sense RNA for chlorophyll a/b bindingprotein to reduce plant chlorophyll content), and 5,741,684 (regulatingthe fertility of pollen in various plants through the use of anti-senseRNA to genes involved in pollen development or function).

Suppression of endogenous thioredoxin or NTR gene expression can also beachieved using ribozymes. Ribozymes are synthetic RNA molecules thatpossess highly specific endoribonuclease activity. The production anduse of ribozymes are disclosed in U.S. Pat. No. 4,987,071 to Cech andU.S. Pat. No. S,543,508 to Haselhoff. Inclusion of ribozymes sequenceswithin antisense RNAs may be used to confer RNA cleaving activity on theantisense RNA, such that endogenous mRNA molecules that bind to theantisense RNA are cleaved, leading to an enhanced antisense inhibitionof endogenous gene expression.

In another embodiment, constructs from which a BTRXh or NTR encodingsequence (or a variant thereof) is overexpressed may be used to obtainco-suppression of the endogenous thioredoxin gene in the mannerdescribed in U.S. Pat. No. 5,231,021 to Jorgensen. Such co-suppression(also termed sense suppression) does not require that the entire BTRXhor NTR encoding sequence be introduced into the plant cells, nor does itrequire that the introduced sequence be exactly identical to theendogenous thioredoxin gene. However, as with antisense suppression, thesuppressive efficiency is enhanced as (1) the introduced sequence islengthened and (2) the sequence similarity between the introducedsequence and the endogenous thioredoxin h gene is increased.

In another embodiment, constructs expressing an untranslatable form of aBTRXh or NTR message may also be used to suppress the expression ofendogenous thioredoxin or NTR activity. Methods for producing suchconstructs are described in U.S. Pat. No. 6,583,021 to Dougherty.Preferably, such constructs are made by introducing a premature stopcodon into the BTRXh or NTR ORF.

Methods of introducing exogenous nucleic acids into a plant host orplant host cells are known in the art. Accordingly, the transformationvector is introduced into plant cells by one of a number of techniques(e.g., electroporation) and progeny plants containing the introducednucleic acid molecule are selected. Preferably all or part of thetransformation vector will stably integrate into the genome of the plantcell. The part of the transformation vector that integrates into theplant cell, and which contains the introduced encoding sequence andassociated expression controlling sequences (the introduced“transgene”), may be referred to as the recombinant expression cassette.

Selection of progeny plants containing the introduced transgene may bemade based upon the detection of an altered phenotype. Such a phenotypemay result directly from the expressed encoding sequence cloned into thetransformation vector (for instance, altered thioredoxin h expression)or may be manifested as enhanced resistance to a chemical agent (such asan antibiotic) as a result of the inclusion of a dominant selectablemarker gene incorporated into the transformation vector.

Successful examples of the modification of plant characteristics bytransformation with cloned cDNA sequences are replete in the technicaland scientific literature. Selected examples, which serve to illustratethe knowledge in this field of technology include:

U.S. Pat. No. 5,571,706 (“Plant Virus Resistance Gene and Methods”)

U.S. Pat. No. 5,677,175 (“Plant Pathogen Induced Proteins”)

U.S. Pat. No. 5,750,386 (“Pathogen-Resistant Transgenic Plants”)

U.S. Pat. No. 5,597,945 (“Plants Genetically Enhanced for Resistance”)

U.S. Pat. No. 5,589,615 (“Process for the Production of TransgenicPlants with Increased Nutritional Value Via the Expression of Modified2S Storage Albumins”)

U.S. Pat. No. 5.750,871 (“Transformation and Foreign Gene Expression inBrassica Species”)

U.S. Pat. No. 5,268,526 (“Over-expression of Phytochrome in TransgenicPlants”)

U.S. Pat. No. 5,780,708 (“Fertile Transgenic Corn Plants”)

U.S. Pat. No. 5,538,880 (“Method For Preparing Fertile Transgenic CornPlants”)

U.S. Pat. No. 5,773,269 (“Fertile Transgenic Oat Plants”)

U.S. Pat. No. 5,736,369 (“Method For Producing Transgenic CerealPlants”)

U.S. Pat. No. 5,610,042 (“Methods For Stable Transformation of Wheat”)

U.S. Pat. No. 5,780,709 (“Transgenic Maize with Increased MannitolContent”)

PCT publication WO 98/48613 (“Compositions and Methods for PlantTransformation and Regeneration”).

These examples include descriptions of transformation vector selection,transformation techniques and the construction of vectors designed toexpress, over-express, or under-express the introduced nucleic acidmolecule. In light of the foregoing and the provision herein of theBTRXh or NTR encoding sequence, it is thus apparent that one of skill inthe art will be able to introduce these nucleic acid sequences, orhomologous or derivative forms of this molecule, into plant cells inorder to produce plants having altered or enhanced barley thioredoxinactivity.

Transformation and regeneration of both monocotyledonous anddicotyledonous plant cells is routine in the art and the practitionerwill determine the appropriate transformation technique. The choice ofmethod will vary with the type of plant to be transformed; those skilledin the art will recognize the suitability of particular methods forgiven plant types. Suitable methods may include, but are not limited to:electroporation of plant protoplasts; liposome-mediated transformation;polyethylene glycol (PEG) mediated transformation; transformation usingviruses; micro-injection of plant cells; micro-injectile bombardment ofplant cells; vacuum infiltration; and Agrobacterium tumefaciens(AT)-mediated transformation. Typical procedures for transforming andregenerating plants are described in the patent documents listed at thebeginning of this section. In addition, certain developmentsparticularly enhance regeneration techniques for monocot plants (see,for instance, U.S. Pat. Nos. 4,666,844 and 5,589,617, and PCTapplication WO 98/48613). For instance, a vector comprising a barleythioredoxin h-encoding nucleic acid can be stably introduced to barleyplants as described in a number of published protocols, including Wanand Lemaux 1994; Lemaux et al., 1996; and Cho et al., 1998a-c).

Depending on the transformation and regeneration protocol followed,transformed plants may be selected using a dominant selectable markerincorporated into the transformation vector or carried on a companionvector used for co-transformation. Typically, such a marker will conferantibiotic or herbicide resistance on the seedlings of transformedplants, and selection of transformants can be accomplished by exposingthe seedlings to appropriate concentrations of the antibiotic.

After transformed plants are selected and grown to maturity, they can beassayed using the methods described herein to determine whetherexpression of thioredoxin h has been altered as a result of theintroduced transgene. Expression of the transformed barley thioredoxin hprotein can be determined by Western blot analysis of transformed planttissues or extracts using standard procedures. BTRXh and NTR activityassays, as discussed above, can be used to determine the activity of theexpressed transgenic Btrwx or NTR. Untransformed and negative segregantplants also are preferably assayed for activity so the background levelof BTRXh or NTR activity (provided by expression of endogenous genes,when present and being expressed) can be determined.

In a preferred embodiment, the BTRXh or NTR proteins are expressed inmammalian cells. Mammalian expression systems are also known in the art,and include retroviral systems. A mammalian promoter is any DNA sequencecapable of binding mammalian RNA polymerase and initiating thedownstream (3′) transcription of a coding sequence for BTRXh or NTRprotein into mRNA. A promoter will have a transcription initiatingregion, which is usually placed proximal to the 5′ end of the codingsequence, and a TATA box, usually located about 25-30 base pairsupstream of the transcription initiation site. The TATA box is thoughtto direct RNA polymerase II to begin RNA synthesis at the correct site.A mammalian promoter will also contain an upstream promoter element(enhancer element), typically located within 100 to 200 base pairupstream of the TATA box. An upstream promoter element determines therate at which transcription is initiated and can act in eitherorientation. Of particular use as mammalian promoters are the promotersfrom mammalian viral genes, since the viral genes are often highlyexpressed and have a broad host range. Examples include the SV40 earlypromoter, mouse mammary tumor virus LTR promoter, adenovirus major latepromoter, herpes simplex virus promoter, and the CMV promoter.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-translational cleavage and polyadenylation.Examples of transcription terminator and polyadenylation signals includethose derived form SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts,as well as other hosts, is well known in the art, and will vary with thehost cell used. Techniques include dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, viral infection, encapsulation ofthe polynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

In a preferred embodiment, BTRXh or NTR proteins are expressed inbacterial systems. Bacterial expression systems are well known in theart.

A suitable bacterial promoter is any nucleic acid sequence capable ofbinding bacterial RNA polymerase and initiating the downstream (3′)transcription of the coding sequence of BTRXh or NTR protein into mRNA.A bacterial promoter has a transcription initiation region which isusually placed proximal to the 5′ end of the coding sequence. Thistranscription initiation region typically includes an RNA polymerasebinding site and a transcription initiation site. Sequences encodingmetabolic pathway enzymes provide particularly useful promotersequences. Examples include promoter sequences derived from sugarmetabolizing enzymes, such as galactose, lactose and maltose, andsequences derived from biosynthetic enzymes such as tryptophan.Promoters from bacteriophage may also be used and are known in the art.In addition, synthetic promoters and hybrid promoters are also useful;for example, the tac promoter is a hybrid of the trp and lac promotersequences. Furthermore, a bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription.

In addition to a functioning promoter sequence, an efficient ribosomebinding site is desirable. In E. coli, the ribosome binding site iscalled the Shine-Delgarno (SD) sequence and includes an initiation codonand a sequence about 3-9 nucleotides in length located about 3-11nucleotides upstream of the initiation codon.

The expression vector may also include a signal peptide sequence thatprovides for secretion of the BTRXh or NTR protein in bacteria. Thesignal sequence typically encodes a signal peptide comprised ofhydrophobic amino acids which direct the secretion of the protein fromthe cell, as is well known in the art. The protein is either secretedinto the growth media (gram-positive bacteria) or into the periplasmicspace, located between the inner and outer membrane of the cell(gram-negative bacteria).

The bacterial expression vector may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed. Suitable selection genes include genes which render thebacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin, kanamycin, neomycin and tetracycline. Selectable markersalso include biosynthetic genes, such as those in the histidine,tryptophan and leucine biosynthetic pathways.

These components are assembled into expression vectors. Expressionvectors for bacteria are well known in the art, and include vectors forBacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcuslividans, among others.

The bacterial expression vectors are transformed into bacterial hostcells using techniques well known in the art, such as calcium chloridetreatment, electroporation, and others.

In one embodiment, BTRXh or NTR proteins are produced in insect cells.Expression vectors for the transformation of insect cells, and inparticular, baculovirus-based expression vectors, are well known in theart.

In a preferred embodiment, BTRXh or NTR protein is produced in yeastcells. Yeast expression systems are well known in the art, and includeexpression vectors for Saccharomyces cerevisiae, Candida albicans and C.maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis,Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, andYarrowia lipolytica. Preferred promoter sequences for expression inyeast include the inducible GAL1,10 promoter, the promoters from alcoholdehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, andacid phosphatase genes. Yeast selectable markers include ADE2, HIS4,LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; theneomycin phosphotransferase gene, which confers resistance to G418; andthe CUP1 gene, which allows yeast to grow in the presence of copperions.

The BTRXh or NTR protein may also be made as a fusion protein, usingtechniques well known in the art. Thus, for example, for the creation ofmonoclonal antibodies, if the desired epitope is small, the BTRXh or NTRprotein may be fused to a carrier protein to form an immunogen.Alternatively, the BTRXh or NTR may be made as a fusion protein toincrease expression, or for other reasons. For example, when the BTRXhor NTR protein is an H. vulgare BTRXh or NTR peptide, the nucleic acidencoding the peptide may be linked to another nucleic acid forexpression purposes. Similarly, BTRXh or NTR proteins of the inventioncan be linked to protein labels, such as green fluorescent protein(GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP),yellow fluorescent protein (YFP), etc.

In one embodiment, the BTRXh or NTR nucleic acids, proteins andantibodies of the invention are labeled. By “labeled” herein is meantthat a compound has at least one element, isotope or chemical compoundattached to enable the detection of the compound. In general, labelsfall into three classes: a) isotopic labels, which may be radioactive orheavy isotopes; b) immune labels, which may be antibodies or antigens;and c) colored or fluorescent dyes. The labels may be incorporated intothe compound at any position.

In a preferred embodiment, the BTRXh or NTR protein is purified orisolated after expression. BTRXh or NTR proteins may be isolated orpurified in a variety of ways known to those skilled in the artdepending on what other components are present in the sample. Standardpurification methods include electrophoretic, molecular, immunologicaland chromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography, and chromatofocusing.For example, the BTRXh or NTR protein may be purified using a standardan antibody column. Ultrafiltration and diafiltration techniques, inconjunction with protein concentration, are also useful. For generalguidance in suitable purification techniques, see Scopes, R., ProteinPurification, Springer-Verlag, NY (1982). The degree of purificationnecessary will vary depending on the use of the BTRXh or NTR protein. Insome instances no purification will be necessary.

Once expressed and purified, if necessary, the BTRXh or NTR proteins andnucleic acids are useful in a number of applications.

The nucleotide sequences (or their complement) encoding BTRXh or NTRproteins have various applications in the art of molecular biology,including uses as hybridization probes, in chromosome and gene mappingand in the generation of anti-sense RNA and DNA. BTRXh or NTR proteinnucleic acid will also be useful for the preparation of BTRXh or NTRproteins by the recombinant techniques described herein.

The full-length native sequence BTRXh or NTR protein gene, or portionsthereof, may be used as hybridization probes for a cDNA library toisolate other genes (for instance, those encoding naturally-occurring,for example, allelic variants of BTRXh or NTR protein or BTRXh or NTRproteins from other genus or species) which have a desired sequenceidentity to the BTRXh or NTR protein coding sequence. Optionally, thelength of the probes will be about 20 to about 50 bases. Thehybridization probes may be derived from the nucleotide sequences hereinor from genomic sequences including promoters, enhancer elements andintrons of native sequences as provided herein. By way of example, ascreening method will comprise isolating the coding region of the BTRXhor NTR protein gene using the known DNA sequence to synthesize aselected probe of about 40 bases. Hybridization probes may be labeled bya variety of labels, including radionuclides such as ³²P or ³⁵S, orenzymatic labels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the BTRXh or NTR protein gene of the presentinvention can be used to screen libraries of human cDNA, genomic DNA ormRNA to determine to which members of such libraries the probehybridizes.

Nucleotide sequences encoding a BTRXh or NTR protein can also be used toconstruct hybridization probes for mapping the gene which encodes thatBTRXh or NTR protein and for the genetic analysis of individuals withgenetic disorders. The nucleotide sequences provided herein may bemapped to a chromosome and specific regions of a chromosome using knowntechniques, such as in situ hybridization, linkage analysis againstknown chromosomal markers, and hybridization screening with libraries.

Nucleic acids which encode BTRXh or NTR protein or its modified formscan also be used to generate either transgenic plants, preferably asdescribed above and in the Examples.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, microparticle bombardment (biolistic)etc. The currently preferred in vivo gene transfer techniques includetransfection with viral (typically retroviral) vectors and viral coatprotein-liposome mediated transfection (Dzau et al., Trends inBiotechnology 11, 205-210 [1993]). In some situations it is desirable toprovide the nucleic acid source with an agent that targets the targetcells to be transformed, such as an antibody specific for a cell surfacemembrane protein or the target cell, a ligand for a receptor on thetarget cell, etc. Where liposomes are employed, proteins which bind to acell surface membrane protein associated with endocytosis may be usedfor targeting and/or to facilitate uptake, e.g. capsid proteins orfragments thereof tropic for a particular cell type, antibodies forproteins which undergo internalization in cycling, proteins that targetintracellular localization and enhance intracellular half-life. Thetechnique of receptor-mediated endocytosis is described, for example, byWu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al.,Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). For review of genemarking and gene therapy protocols see Anderson et al., Science 256,808-813 (1992).

In a preferred embodiment, the BTRXh or NTR proteins, nucleic acids,variants, modified proteins, cells and/or transgenics containing thesaid nucleic acids or proteins are used in screening assays.Identification of the BTRXh or NTR protein provided herein permits thedesign of screening assays for compounds that bind or modulate BTRXh orNTR activity.

“Modulating the activity of a BTRXh or NTR protein” includes an increasein activity, a decrease in activity, or a change in the type or kind ofactivity present. Thus, in this embodiment, the candidate agent shouldboth bind to BTRXh or NTR protein (although this may not be necessary),and alter its biological or biochemical activity as defined herein. Themethods include both in vitro screening methods, as are generallyoutlined above, and in vivo screening of cells for alterations in thepresence, distribution, activity or amount of the BTRXh or NTR protein.

Methods of assaying a biological activity of a thioredoxin h protein areknown in the art. Thioredoxins lack a directly measurable catalyticproperty that can be used in their detection and quantification.Therefore, a specific companion enzyme reaction is necessary todemonstrate and measure the activity of thioredoxin proteins. One ofordinary skill in the art will be aware that there are manywell-established systems that can be employed to measurethioredoxin-mediated reduction of substrates. See, for instance, U.S.Pat. No. 5,792,506 to Buchanan; Horecka et al. (1996); Rivera-Madrid etal. (1995); Wong et al. (1995); Jacquot et al. (1990); Florencio et al.(1988); Florencio et al. (1988); Johnson et al. (1987); Schwenn andSchriek (1986); and Berstermann et al. (1983). Appropriate assay systemsmay be broken down into four major classes: 1) enzyme activation (usingthioredoxin to modulate another enzyme in a measurable way); 2)ribonucleotide reduction (using thioredoxin as a co-substrate byribonucleotide reductase); 3) protein disulfide reduction (usingthioredoxin for insulin A-B chain reduction) (Schwenn and Schriek,1987); and 4) direct measurement of disulfide reduction usingmonobromobimane (mBBr)-derivative fluorescence (Wong et al., 1995). Anysuch system can be used to measure thioredoxin activity. Differenttechniques will be more or less appropriate to specific plants andtissues. The appropriate technique will be determined at the discretionof the practitioner. By way of example, the following techniques areappropriate for measuring the activity of thioredoxin.

Because of interference from other enzymes that use NADPH, the activityof thioredoxin h cannot be accurately assayed in crude plant extracts.Thus, at least partial purification of the thioredoxin h protein isnecessary. Such partial purification can be carried out using standardprotein purification techniques, for instance (NH₄)₂SO₄ extraction andcolumn chromatography. See, for example, Florencio et al. (1988) andJohnson et al. (1987a,b).

Thioredoxin h activity-assayable transgenic barley seed extracts can beprepared in the following manner. In the embodiment, wherein Btrwhactivity is assayed in a plant or plant tissue, such as a seed or grain,about fifteen grams of barley grains, may be ground to powder in acoffee grinder or other like device. Protein is subsequently extractedfrom this powder with 80 ml (1:4 w/v) of buffer (50 mM Tris-HCl buffer,pH 7.0, 1 mM EDTA, 0.5 mM PMSF (phenylmethysulfonyl fluoride). 2 mMe-amino-n caproic acid, 2 mM benzamidine-HCl) by stirring for threehours at 4° C. The slurry plus the rinse is then subjected tocentrifugation at 25,400×g for 20 minutes, and the resultant supernatantsolution decanted through glass wool. The pellet is resuspended in asmall volume of buffer and then clarified by centrifugation as before.The two supernatant fractions are combined, an aliquot removed as acontrol, and the remainder subjected to acidification by adjusting thepH from about 7.83 to about 4.80 with 2 N formic acid. Denaturedproteins should be removed from the acidified solution by centrifugationas above prior to assaying for enzyme activity. The pH of the acidifiedsupernatant solution is then readjusted to 7.91 with 2 N NH₄OH and analiquot is removed for enzyme assay. Powdered (NH₄)₂SO₄ is added to afinal concentration of 30% (w/v) and the sample stirred for 20 minutesat 4° C., followed by centrifugation as described above. Additional(NH₄)₂SO₄ is then added to bring the decanted supernatant solution to90% (w/v) saturation, and the sample stirred for 16 hours at 4° C. Thissample is centrifuged again as described above to yield a thioredoxinh-enriched pellet.

The supernatant solution from the thioredoxin h-enriched pellet isdiscarded, and the pellet re-suspended in 30 mM Tris-HCl, pH 7.9 buffer.This is then clarified by centrifugation at 40,000×g for 15 minutes. Theresulting supernatant (30-50% (NH₄)₂SO₄ fraction) should then be placedin dialysis tubing (6,000-8,000 MW cut-off) and exposed to solid sucroseat 4° C. to obtain an approximate 10-fold reduction in volume. Analiquot (about 1 ml) of the clarified and concentrated 30-90% (NH₄)₂SO₄sample should be reserved. The remaining sample is applied to a preequilibrated (30 mM Tris-HCl, pH 7.9, 200 mM NaCl) Sephadex G-50superfine column (2.5×90 cm; ˜400 ml bed volume) with a peristaltic pumpat a flow rate of 0.5 ml/min. Protein is eluted from the loaded columnwith the same buffer at the same flow rate, and 150-drop fractionscollected. Each fraction can be tested for thioredoxin h activity usingstandard techniques, for instance the NADP-MDH activation protocol (seebelow). Storage of the prepared fractions should be at 4° C.

Thioredoxin h extracted from E. coli is stable after treatment at 60° C.for 10 minutes (Mark and Richardson, 1976). Using this feature ofthioredoxin h proteins, the level of background (non-thioredoxin h)enzyme activity in crude plant extracts can be decreased by heating thecrude extract at 60° C. for about ten minutes, using the followingprotocol. In the example of expression of BTRXh in transgenic grain,approximately ten grams of, for example, barley grain are ground topowder for about 30 seconds in a coffee grinder and extracted by shakingfor 1 hour at room temperature in 50 ml buffer (50 mM Tris-HCl buffer,pH 7.9. 1 mM EDTA, 0.5 mM PMSF, 2 mM e-amino-n caproic acid, 2 mMbenzamidine-HCl). The slurry plus the rinse is then subjected tocentrifugation at 27,000×g for 20 minutes and the supernatant solutiondecanted through glass wool. A 20 ml aliquot of the supernatant is thenheated at 65° C. until sample temperature reaches 60+1° C. (˜10minutes). The sample is then held at 60° C. for 10 additional minutes,then cooled in an ice/water bath. The cooled sample is centrifuged andthe supernatant solution concentrated using solid sucrose as above. Theresultant heat-treated, concentrated supernatant may be stored at −20°C. Frozen samples are thawed and clarified by centrifugation at 14,000rpm for 10 minutes at 4° C. Total thioredoxin h activity can then bemeasured in these concentrated supernatant fractions.

Methods and techniques of measuring thioredoxin h activity havepreviously been described (see, for instance, Berstermann et al. 1983;Johnson et al. 1987; and Florencio et al. 1988). For each technique,about fifty to 120 μl (depending on activity) of partially purified orheat-treated plant extract as prepared above is pre-incubated with DTT,and 0.16 to 0.32 μl of this pre-incubation mixture is then used in theassay.

In general, thioredoxin h activity is assayed by addingthioredoxin-bearing sample to an NADP-thioredoxin reductase (NTR) assaysystem (Florencio et al., 1988; Gautier et al, 1998), and the reductionof DTNB measured. Essentially, NADPH provides the reducing equivalentsneeded for thioredoxin reductase to reduce thioredoxin h by convertingit from the disulfide (—S—S—) to the sulthydryl (—SH) form. This reduced(sulfhydryl) thioredoxin h then reduces DTNB directly. Reduction of DTNBis measured as an increase in absorbance of the sample at 412 nm.

By way of example, 1 ml reaction mixtures containing 100 μM potassiumphosphate (pH 7.1), 10 μl mol @@@ 150 nmol EDTA, 150 nmol NADPH, 200nmol DTNB (dissolved in 95% ethanol) and variable amounts of thioredoxinh-bearing samples are initiated by the addition of 10 pmol of wheat orE. coli NTR, and the reduction of DTNB determined by monitoring theabsorbance change at 412 nm. The activity can then be expressed as μmolthioredoxin reduced per minute using 13,600 M⁻¹ cm⁻¹ as the molarabsorption coefficient of DTNB (2-SH being formed/mol reducedthioredoxin).

In the NADP-Malate Dehydrogenase (MDH) Activation Assay, thioredoxin hactivity is assayed by adding thioredoxin-bearing sample to anNADP-malate dehydrogenase (MDH) assay system (Johnson et al., 1987;Berstermann et al. 1983). This system is similar to that used in anNADP-NTR activation assay.

In vitro monobromobimane (mBBr) labeling of proteins is an alternate toindirectly measuring thioredoxin h protein activity using acompanion-enzyme assay, thioredoxin-mediated disulfide reduction can bemeasured using monobromobimane (mBBr) derivative fluorescence (Crawfordet al., 1989; Kobrehel et al., 1992; U.S. Pat. No. 5,792,506“Neutralization of food allergens by thioredoxin”). By way of exampleonly, the following describes an appropriate procedure as it relates totransgenic plants. Immature, mature, or germinating seeds or grain fromnontransformed (control) and transgenic plants are ground in 100 mMTris-HCl buffer, pH 7.9. Assay reactions may then be carried outessentially as described in Kobrehel et al. (1992). In general, 70 μL ofthe buffer mixture containing a known amount of protein is eitheruntreated or treated with DTT to a final concentration of 0.5 mM. Afterincubation for 20 minutes, 100 nmol of mBBr is added, and the reactioncontinued for another 15 minutes. To stop the reaction and derivatizeexcess mBBr, 10 μl of 10% SDS and 100 μl of 100 mM 2-mercaptoethanol areadded. The samples are then applied to a 15% SDS-PAGE gel. Fluorescenceof mBBr may be visualized by placing gels on a light box fitted with aUV (365 nm) light source. Protein quantification can be carried out bythe Bradford dye-binding method (Bradford, 1976) using, for instance,bovine serum albumin or gamma globulin as standards. This protocol hasbeen adapted for barley as described by Cho et al, ((1999) Proc. Natl.Acad. Sci. USA 96:14641-14646).

Methods of measuring NTR activity also are known in the art. In apreferred embodiment, NTR activity is determined with the DNTB assay(Florencio et al., 1988). The system contains the amount of an extract,as needed, spinach thioredoxin h (2-5 μg) and the following 100 μmolpotassium phosphate buffer (pH 7.9). Ten μmol Na-EDTA; 0.25 μmol NADPH,0.2 μmol DTNB. The reaction is started by the addition of thioredoxin h(final volume, 1.0 ml). Increase in absorbance is followed at 412 nm.

The content and activity of BTRXh and NTR is alternatively assessed byWestern blot and activity measurements. In the example of transgenicseeds or grains, western blots are performed on extracts selectedtransgenic seeds or grain as well as non-transgenic seeds or grains,including null segregants. Lots of about 10-20 intacts seeds are grainsare processed and analyzed for content of BTRXh end NTR by SDS-PAGE andwestern blot procedures (Cho et al, (1999) Proc. Natl. Acad. Sci. USA96:14641-14646). Grain or seeds extract are preferably prepared asdescribed by Cho et al., (1999) Plant Sci. 148:9-17).

Thus, in the embodiment of identifying a bioactive agent the alters abiological activity of a BTRXh or NTR polypeptide, the methods comprisecombining an BTRXh or NTR sample and a candidate bioactive agent, andevaluating the effect on the BTRXh or NTR activity. By “BTRXh or NTRactivity” or grammatical equivalents herein is meant one of the BTRXh orNTR protein's biological activities, including, but not limited to thosedescribed above and, for example, a BTRXh or NTR proteins ability toalter the oxidation/reduction state of NADPH₂ or thioredoxin in vitro orin vivo. Other biological activities include altering theoxidation/reduction state of a cell of a transgenic plant in which it isexpressed, altering the digestibility of the starch or proteincomponents of a transgenic seed or grain; redistribution of the proteinsof a transgenic seed or grain to the more soluble albumin/globulinfraction and decreasing the allergenicity of a transgenic seed or grain.

In a preferred embodiment, the activity of the BTRXh or NTR protein isdecreased; in another preferred embodiment, the activity of the BTRXh orNTR protein is increased. Thus, bioactive agents that are antagonistsare preferred in some embodiments, and bioactive agents that areagonists may be preferred in other embodiments.

The assays described herein preferably utilize the H. vulgare BTRXh orNTR protein, although other plant proteins may also be used, includingdicotyledonous plants (e.g. tomato, potato, soybean, cotton, tobacco,etc.) and monocotyledonous plants, including, but not limited tograminaceous monocots such as wheat (Triticum spp.), rice (Oryza spp.),barley (Hordeum spp.), oat (Avena spp.), rye (Secale spp.), corn (Zeamays), sorghum (Sorghum spp.) and millet (Pennisetum spp). For example,the present invention can be employed with barley genotypes including,but not limited to Morex, Harrington, Crystal, Stander, Moravian III,Galena, Salome, Steptoe, Klages, Baronesse, and with wheat genotypesincluding, but not limited to Yecora Rojo, Bobwhite, Karl and Anza. Ingeneral, the invention is particularly useful in cereals. These latterembodiments may be preferred in the development of models of graingermination.

In a preferred embodiment, the methods comprise combining a BTRXh or NTRprotein and a candidate bioactive agent, and determining the binding ofthe candidate agent to the BTRXh or NTR protein. In other embodiments,further discussed below, binding interference or bioactivity isdetermined.

The term “candidate bioactive agent” or “exogeneous compound” as usedherein describes any molecule, e.g., protein, small organic molecule,carbohydrates (including polysaccharides), polynucleotide, lipids, etc.Generally a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a differential response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e., at zero concentration or below the level ofdetection. In addition, positive controls, i.e. the use of agents knownto alter or modulate BTRXh or NTR activity, may be used.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than about 100 daltons and less than about2,500 daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof. Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

In a preferred embodiment, a library of different candidate bioactiveagents are used. Preferably, the library should provide a sufficientlystructurally diverse population of randomized agents to effect aprobabilistically sufficient range of diversity to allow binding to aparticular target. Accordingly, an interaction library should be largeenough so that at least one of its members will have a structure thatgives it affinity for the target. Although it is difficult to gauge therequired absolute size of an interaction library, nature provides a hintwith the immune response: a diversity of 10⁷-10⁸ different antibodiesprovides at least one combination with sufficient affinity to interactwith most potential antigens faced by an organism. Published in vitroselection techniques have also shown that a library size of 10⁷ to 10⁸is sufficient to find structures with affinity for the target. A libraryof all combinations of a peptide 7 to 20 amino acids in length, such asgenerally proposed herein, has the potential to code for 20⁷ (10⁹) to20²⁰. Thus, with libraries of 10⁷ to 10⁸ different molecules the presentmethods allow a “working” subset of a theoretically complete interactionlibrary for 7 amino acids, and a subset of shapes for the 20²⁰ library.Thus, in a preferred embodiment, at least 10⁶, preferably at least 10⁷,more preferably at least 10⁸ and most preferably at least 10⁹ differentsequences are simultaneously analyzed in the subject methods. Preferredmethods maximize library size and diversity.

In a preferred embodiment, the candidate bioactive agents are proteins.By “protein” herein is meant at least two covalently attached aminoacids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chains may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations. Chemical blocking groups orother chemical substituents may also be added.

In a preferred embodiment, the candidate bioactive agents are naturallyoccurring proteins or fragments of naturally occurring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. Inthis way libraries of procaryotic and eukaryotic proteins may be madefor screening in the systems described herein. Particularly preferred inthis embodiment are libraries of plant, bacterial, fungal, viral, andmammalian proteins, with the latter being preferred, and human proteinsbeing especially preferred.

In a preferred embodiment, the candidate bioactive agents are peptidesof from about 5 to about 30 amino acids, with from about 5 to about 20amino acids being preferred, and from about 7 to about 15 beingparticularly preferred. The peptides may be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. By “randomized” or grammatical equivalents herein ismeant that each nucleic acid and peptide consists of essentially randomnucleotides and amino acids, respectively. Since generally these randompeptides (or nucleic acids, discussed below) are chemically synthesized,they may incorporate any nucleotide or amino acid at any position. Thesynthetic process can be designed to generate randomized proteins ornucleic acids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the candidate bioactive agents are nucleicacids. By “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein means at least two nucleotides covalently linked together. Anucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925(1993) and references therein; Letsinger, J. Org. Chem., 35:3800 (1970);Sprinzl, et al., Eur. J. Biochem., 81:579 (1977); Letsinger, et al.,Nucl. Acids Res., 14:3487 (1986); Sawai, et al., Chem. Lett. 805 (1984),Letsinger, et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels, etal., Chemica Scripta, 26:141 (1986)), phosphorothioate (Mag, et al.,Nucleic Acids Res., 19:1437 (1991); and U.S. Pat. No. 5,644,048),phosphorodithioate (Briu, et al., J. Am. Chem. Soc., 111:2321 (1989)),O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), and peptidenucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc.,114:1895 (1992); Meier, et al., Chem. Int. Ed. Engl., 31:1008 (1992);Nielsen, Nature, 365:566 (1993); Carlsson, et al., Nature, 380:207(1996), all of which are incorporated by reference)). Other analognucleic acids include those with positive backbones (Denpcy, et al.,Proc. Natl. Acad. Sci. USA, 92:6097 (1995)); non-ionic backbones (U.S.Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141; and 4,469,863;Kiedrowshi, et al., Angew. Chem. Intl. Ed. English, 30:423 (1991);Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988); Letsinger, etal., Nucleoside & Nucleotide, 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed Y. S. Sanghui and P. Dan Cook; Mesmaeker, et al.,Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J.Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins,et al., Chem. Soc. Rev., (1995) pp. 169-176). Several nucleic acidanalogs are described in Rawls, C & E News, Jun. 2, 1997, page 35. Allof these references are hereby expressly incorporated by reference.These modifications of the ribose-phosphate backbone may be done tofacilitate the addition of additional moieties such as labels, or toincrease the stability and half-life of such molecules in physiologicalenvironments. In addition, mixtures of naturally occurring nucleic acidsand analogs can be made. Alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made. The nucleic acids may be single stranded or doublestranded, as specified, or contain portions of both double stranded orsingle stranded sequence. The nucleic acid may be DNA, both genomic andcDNA, RNA or a hybrid, where the nucleic acid contains any combinationof deoxyribo- and ribo-nucleotides, and any combination of bases,including uracil, adenine, thymine, cytosine, guanine, inosine,xathanine hypoxathanine, isocytosine, isoguanine, etc.

As described above generally for proteins, nucleic acid candidatebioactive agents may be naturally occurring nucleic acids, randomnucleic acids, or “biased” random nucleic acids. For example, digests ofprocaryotic or eukaryotic genomes may be used as is outlined above forproteins.

In a preferred embodiment, the candidate bioactive agents are organicchemical moieties, a wide variety of which are available in theliterature.

In a preferred embodiment, the candidate bioactive agents are linked toa fusion partner. By “fusion partner” or “functional group” herein ismeant a sequence that is associated with the candidate bioactive agent,that confers upon all members of the library in that class a commonfunction or ability. Fusion partners can be heterologous (i.e. notnative to the host cell), or synthetic (not native to any cell).Suitable fusion partners include, but are not limited to: a)presentation structures, which provide the candidate bioactive agents ina conformationally restricted or stable form; b) targeting sequences,which allow the localization of the candidate bioactive agent into asubcellular or extracellular compartment; c) rescue sequences whichallow the purification or isolation of either the candidate bioactiveagents or the nucleic acids encoding them; d) stability sequences, whichconfer stability or protection from degradation to the candidatebioactive agent or the nucleic acid encoding it, for example resistanceto proteolytic degradation; e) dimerization sequences, to allow forpeptide dimerization; or f) any combination of a), b), c), d), and e),as well as linker sequences as needed.

In one embodiment of the methods described herein, portions of BTRXh orNTR proteins are utilized; in a preferred embodiment, portions havingBTRXh or NTR activity are used to identify agents that bind to BTRXh orNTR. In addition, the assays described herein may utilize eitherisolated BTRXh or NTR proteins or cells comprising the BTRXh or NTRproteins.

Generally, in a preferred embodiment of the methods herein, for examplefor binding assays, the BTRXh or NTR protein or the candidate agent isnon-diffusibly bound to an insoluble support having isolated samplereceiving areas (e.g. a microtiter plate, an array, etc.). The insolublesupports may be made of any composition to which the compositions can bebound, is readily separated from soluble material, and is otherwisecompatible with the overall method of screening. The surface of suchsupports may be solid or porous and of any convenient shape. Examples ofsuitable insoluble supports include microtiter plates, arrays, membranesand beads. These are typically made of glass, plastic (e.g.,polystyrene), polysaccharides, nylon or nitrocellulose, teflon™, etc.Microtiter plates and arrays are especially convenient because a largenumber of assays can be carried out simultaneously, using small amountsof reagents and samples. In some cases magnetic beads and the like areincluded. The particular manner of binding of the composition is notcrucial so long as it is compatible with the reagents and overallmethods of the invention, maintains the activity of the composition andis nondiffusable. Preferred methods of binding include the use ofantibodies (which do not sterically block either the ligand binding siteor activation sequence when the protein is bound to the support), directbinding to “sticky” or ionic supports, chemical crosslinking, thesynthesis of the protein or agent on the surface, etc. Following bindingof the protein or agent, excess unbound material is removed by washing.The sample receiving areas may then be blocked through incubation withbovine serum albumin (BSA), casein or other innocuous protein or othermoiety. Also included in this invention are screening assays whereinsolid supports are not used; examples of such are described below.

In a preferred embodiment, the BTRXh or NTR protein is bound to thesupport, and a candidate bioactive agent is added to the assay.Alternatively, the candidate agent is bound to the support and the BTRXhor NTR protein is added. Novel binding agents include specificantibodies, non-natural binding agents identified in screens of chemicallibraries, peptide analogs, etc. Of particular interest are screeningassays for agents that have a low toxicity for human cells. A widevariety of assays may be used for this purpose, including labeled invitro protein-protein binding assays, electrophoretic mobility shiftassays, immunoassays for protein binding, functional assays, preferablyoxidation/reduction assays.

The determination of the binding of the candidate bioactive agent to theBTRXh or NTR protein may be done in a number of ways. In a preferredembodiment, the candidate bioactive agent is labelled, and bindingdetermined directly. For example, this may be done by attaching all or aportion of the BTRXh or NTR protein to a solid support, adding alabelled candidate agent (for example a radio or fluorescent label),washing off excess reagent, and determining whether the label is presenton the solid support. Various blocking and washing steps may be utilizedas is known in the art.

By “labeled” herein is meant that the compound is either directly orindirectly labeled with a label which provides a detectable signal, e.g.radioisotope, fluorescers, enzyme, antibodies, particles such asmagnetic particles, chemiluminescers, or specific binding molecules,etc. Specific binding molecules include pairs, such as biotin andstreptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

In some embodiments, only one of the components is labeled. For example,the proteins (or proteinaceous candidate agents) may be labeled attyrosine positions using ¹²⁵I, or with fluorophores. Alternatively, morethan one component may be labeled with different labels; using ¹²⁵I forthe proteins, for example, and a fluorophor for the candidate agents.

In a preferred embodiment, the binding of the candidate bioactive agentis determined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to thetarget molecule (i.e. BTRXh or NTR protein), such as an antibody,peptide, binding partner, ligand, etc. Under certain circumstances,there may be competitive binding as between the bioactive agent and thebinding moiety, with the binding moiety displacing the bioactive agent.This assay can be used to determine candidate agents which interferewith binding between BTRXh or NTR proteins and binding partners.“Interference of binding” as used herein means that native binding ofthe BTRXh or NTR protein differs in the presence of the candidate agent.The binding can be eliminated or can be with a reduced affinity.Therefore, in one embodiment, interference is caused by, for example, aconformation change, rather than direct competition for the nativebinding site.

In one embodiment, the candidate bioactive agent is labeled. Either thecandidate bioactive agent, or the competitor, or both, is added first tothe protein for a time sufficient to allow binding, if present.Incubations may be performed at any temperature which facilitatesoptimal activity, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high through put screening. Typically between about 0.1 and about1.0 hour will be sufficient. Excess reagent is generally removed orwashed away. The second component is then added, and the presence orabsence of the labeled component is followed, to indicate binding.

In a preferred embodiment, the competitor is added first, followed bythe candidate bioactive agent. Displacement of the competitor is anindication that the candidate bioactive agent is binding to the NTRprotein and thus is capable of binding to, and potentially modulating,the activity of the NTR protein. In this embodiment, either componentcan be labeled. Thus, for example, if the competitor is labeled, thepresence of label in the wash solution indicates displacement by theagent. Alternatively, if the candidate bioactive agent is labeled, thepresence of the label on the support indicates displacement.

In an alternative embodiment, the candidate bioactive agent is addedfirst with incubation and washing, followed by the competitor. Theabsence of binding by the competitor may indicate that the bioactiveagent is bound to the BTRXh or NTR protein with a higher affinity. Thus,it the candidate bioactive agent is labeled, the presence of the labelon the support, coupled with a lack of competitor binding, may indicatethat the candidate agent is capable of binding to the BTRXh or NTRprotein.

In a preferred embodiment, the methods comprise differential screeningto identity bioactive agents that are capable of modulating the activityof the BTRXh or NTR proteins. Such assays can be done with the BTRXh orNTR protein or cells comprising said BTRXh or NTR protein. In oneembodiment, the methods comprise combining an BTRXh or NTR protein and acompetitor in a first sample. A second sample comprises a candidatebioactive agent, an BTRXh or NTR protein and a competitor. The bindingof the competitor is determined for both samples, and a change, ordifference in binding between the two samples indicates the presence ofan agent capable of binding to the BTRXh or NTR protein and potentiallymodulating its activity. That is, if the binding of the competitor isdifferent in the second sample relative to the first sample, the agentis capable of binding to the BTRXh or NTR protein.

Alternatively, a preferred embodiment utilizes differential screening toidentify candidates that bind to the native BTRXh or NTR protein, butcannot bind to modified BTRXh or NTR proteins. The structure of theBTRXh or NTR protein may be modeled, and used in rational design andsynthesis of agents that interact with that site. Drug candidates thataffect BTRXh or NTR bioactivity are also identified by screening drugsfor the ability to either enhance or reduce the activity of the protein.

Positive controls and negative controls may be used in the assays.Preferably all control and test samples are performed in at leasttriplicate to obtain statistically significant results. Incubation ofall samples is for a time sufficient for the binding of the agent to theprotein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

In a preferred embodiment, the invention provides methods of screeningfor bioactive agents capable of modulating the activity of an BTRXh orNTR protein. The methods comprise adding a candidate bioactive agent asdefined above, to a cell comprising BTRXh or NTR proteins. Preferredcell types include almost any cell. The cells contain a recombinantnucleic acid that encodes an BTRXh or NTR protein. In a preferredembodiment, a library of candidate agents are tested on a plurality ofcells.

Detection of BTRXh or NTR regulation may be done as will be appreciatedby those in the art. In one embodiment, indicators of the NTR activityare used, for example, oxidation of NADPH or reduction of thioredoxin,preferably thioredoxin h. In one embodiment, indicators of the BTRXhactivity are used, for example, using the NADP-malate dehydrogenaseactivation assay as described by Florencio et al. 1988 and Johnson etal. (1987a). There are a number of parameters that may be evaluated orassayed to allow the detection of alterations in BTRXh or NTRregulation, including, but not limited to, cell viability assays,germination characteristics of a transgenic grain or seed, redox statusof transgenic grain or seed, digestibility of a transgenic seed orgrain, the expression of gibberellic acid inducible enzyme in atransgenic seed or grain. Other parameters include mRNA synthesis,translation, peptides, activity of a protein or enzyme, distribution ofprotein in, for example, more soluble verses less soluble fractions. Byassaying or measuring one or more of these parameters, it is possible todetect not only alterations in BTRXh or NTR regulation, but alterationsof different steps of the BTRXh or NTR regulation pathway. In thismanner, rapid, accurate screening of candidate agents may be performedto identify agents that modulate BTRXh or NTR regulation.

Accordingly, the invention provides methods of screening for alterationsin BTRXh or NTR regulation of a population of cells. By “alteration” or“modulation” (used herein interchangeably), is generally meant a change,for example, in the redox state of a substrate or co-factor of the BTRXhor NTR protein. In another embodiment, is meant a change in the redoxstate in a pathway affected by BTRXh or NTR activity.

The measurements can be determined wherein all of the conditions are thesame for each measurement, or under various conditions, with or withoutbioactive agents, or at different stages of the cell cycle process. Forexample, a measurement of BTRXh or NTR regulation can be determined in acell or cell population wherein a candidate bioactive agent is presentand wherein the candidate bioactive agent is absent. In another example,the measurements of BTRXh or NTR regulation are determined wherein thecondition or environment of the cell or populations of cells differ fromone another. For example, the cells may be evaluated in the presence orabsence or previous or subsequent exposure of physiological signals, forexample hormones, antibodies, peptides, antigens, cytokines, growthfactors, action potentials, pharmacological agents includingchemotherapeutics, radiation, carcinogenics, or other cells (i.e.cell-cell contacts). In another example, the measurements of BTRXh orNTR regulation are determined at different stages of the cell cycleprocess. In yet another example, the measurements of BTRXh or NTRregulation are taken wherein the conditions are the same, and thealterations are between one cell or cell population and another cell orcell population.

By a “population of cells” or “library of cells” herein is meant atleast two cells, with at least about 10³ being preferred, at least about10⁵ being particularly preferred, and at least about 10⁵ to 10⁹ beingespecially preferred. The population or sample can contain a mixture ofdifferent cell types from either primary or secondary cultures althoughsamples containing only a single cell type are preferred, for example,the sample can be from a cell line, particularly tumor cell lines, asoutlined below. The cells may be in any cell phase, either synchronouslyor not, including M, G1, S, and G2. In a preferred embodiment, cellsthat are replicating or proliferating are used; this may allow the useof retroviral vectors for the introduction of candidate bioactiveagents. Alternatively, non-replicating cells may be used, and othervectors (such as adenovirus, lentivirus, and Agrobacterium vectors) canbe used. In addition, although not required, the cells are compatiblewith dyes and antibodies.

Preferred cell types for use in the invention include, but are notlimited to, plant cells, including mono- and dicot plants such as(cereal grains, barley, wheat sorghum, soybeans, sugar beets, peanuts,canola and as described above), and mammalian cells, including animal(rodents, including mice, rats, hamsters and gerbils), primates, andhuman cells, particularly including tumor cells of all types, includingbreast, skin, lung, cervix, colon-rectal, leukemia, brain, etc.

In a preferred embodiment, the methods comprise assaying one or more ofseveral different cell parameters, including, but not limited to, cellviability, cell proliferation, and cell phase.

In a preferred embodiment, cell viability is assayed, to ensure that alack of cellular change is due to experimental conditions (i.e. theintroduction of a candidate bioactive agent) not cell death. There are avariety of suitable cell viability assays which can be used, including,but not limited to, light scattering, viability dye staining, andexclusion dye staining.

In a preferred embodiment, a light scattering assay is used as theviability assay, as is well known in the art. For example, when viewedin the FACS, cells have particular characteristics as measured by theirforward and 90 degree (side) light scatter properties. These scatterproperties represent the size, shape and granule content of the cells.These properties account for two parameters to be measured as a readoutfor the viability. Briefly, the DNA of dying or dead cells generallycondenses, which alters the 90° scatter; similarly, membrane blebbingcan alter the forward scatter. Alterations in the intensity of lightscattering, or the cell-refractive index indicate alterations inviability.

Thus, in general, for light scattering assays, a live cell population ofa particular cell type is evaluated to determine its forward and sidescattering properties. This sets a standard for scattering that cansubsequently be used.

In a preferred embodiment, the viability assay utilizes a viability dye.There are a number of known viability dyes that stain dead or dyingcells, but do not stain growing cells. For example, annexin V is amember of a protein family which displays specific binding tophospholipid (phosphotidylserine) in a divalent ion dependent manner.This protein has been widely used for the measurement of apoptosis(programmed cell death) as cell surface exposure of phosphatidylserineis a hallmark early signal of this process. Suitable viability dyesinclude, but are not limited to, annexin, ethidium homodimer-1, DEADRed, propidium iodide, SYTOX Green, etc., and others known in the art;see the Molecular Probes Handbook of Fluorescent Probes and ResearchChemicals, Haugland, Sixth Edition, hereby incorporated by reference;see Apoptosis Assay on page 285 in particular, and Chapter 16.

Protocols for viability dye staining for cell viability are known, seeMolecular Probes catalog, supra. In this embodiment, the viability dyesuch as annexin is labeled, either directly or indirectly, and combinedwith a cell population. Annexin is commercially available, i.e., fromPharMingen, San Diego, Calif., or Caltag Laboratories, Millbrae, Calif.Preferably, the viability dye is provided in a solution wherein the dyeis in a concentration of about 100 ng/ml to about 500 ng/ml, morepreferably, about 500 ng/ml to about 1 μg/ml, and most preferably, fromabout 1 μg/ml to about 5 μg/ml. In a preferred embodiment, the viabilitydye is directly labeled; for example, annexin may be labeled with afluorochrome such as fluorecein isothiocyanate (FITC), Alexa dyes,TRITC, AMCA, APC, tri-color, Cy-5, and others known in the art orcommercially available. In an alternate preferred embodiment, theviability dye is labeled with a first label, such as a hapten such asbiotin, and a secondary fluorescent label is used, such as fluorescentstrain. Other first and second labeling pairs can be used as will beappreciated by those in the art.

Once added, the viability dye is allowed to incubate with the cells fora period of time, and washed, if necessary. The cells are then sorted asoutlined below to remove the non-viable cells.

In a preferred embodiment, exclusion dye staining is used as theviability assay. Exclusion dyes are those which are excluded from livingcells, i.e. they are not taken up passively (they do not permeate thecell membrane of a live cell). However, due to the permeability of deador dying cells, they are taken up by dead cells. Generally, but notalways, the exclusion dyes bind to DNA, for example via intercalation.Preferably, the exclusion dye does not fluoresce, or fluoresces poorly,in the absence of DNA; this eliminates the need for a wash step.Alternatively, exclusion dyes that require the use of a secondary labelmay also be used. Preferred exclusion dyes include, but are not limitedto, ethidium bromide; ethidium homodimer-1; propidium iodine; SYTOXgreen nucleic acid stain; Calcein AM, BCECF AM; fluorescein diacetate;TOTO® and TO-PRO™ (from Molecular Probes; supra, see chapter 16) andothers known in the art.

Protocols for exclusion dye staining for cell viability are known, seethe Molecular Probes catalog, supra. In general, the exclusion dye isadded to the cells at a concentration of from about 100 ng/ml to about500 ng/ml, more preferably, about 500 ng/ml to about 1 μg/ml, and mostpreferably, from about 0.1 μg/ml, to about 5 μg/ml, with about 0.5 μg/mlbeing particularly preferred. The cells and the exclusion dye areincubated for some period of time, washed, if necessary, and then thecells sorted as outlined below, to remove non-viable cells from thepopulation.

In addition, there are other cell viability assays which may be run,including for example enzymatic assays, which can measure extracellularenzymatic activity of either live cells (i.e. secreted proteases, etc.),or dead cells (i.e. the presence of intracellular enzymes in the media;for example, intracellular proteases, mitochondrial enzymes, etc.). Seethe Molecular Probes Handbook of Fluorescent Probes and ResearchChemicals, Haugland, Sixth Edition, hereby incorporated by reference;see chapter 16 in particular.

In a preferred embodiment, at least one cell viability assay is run,with at least two different cell viability assays being preferred, whenthe fluors are compatible. When only 1 viability assay is run, apreferred embodiment utilizes light scattering assays (both inward andside scattering). When two viability assays are run, preferredembodiments utilize light scattering and dye exclusion, with lightscattering and viability dye staining also possible, and all three beingdone in some cases as well. Viability assays thus allow the separationof viable cells from non-viable or dying cells.

In addition to a cell viability assay, a preferred embodiment utilizes acell proliferation assay. By “proliferation assay” herein is meant anassay that allows the determination that a cell population is eitherproliferating, i.e. replicating, or not replicating.

In a preferred embodiment, the proliferation assay is a dye inclusionassay. A dye inclusion assay relies on dilution effects to distinguishbetween cell phases. Briefly, a dye (generally a fluorescent dye asoutlined below) is introduced to cells and taken up by the cells. Oncetaken up, the dye is trapped in the cell, and does not diffuse out. Asthe cell population divides, the dye is proportionally diluted. That is,after the introduction of the inclusion dye, the cells are allowed toincubate for some period of time; cells that lose fluorescence over timeare dividing, and the cells that remain fluorescent are arrested in anon-growth phase.

Generally, the introduction of the inclusion dye may be done in one oftwo ways. Either the dye cannot passively enter the cells (e.g. it ischarged), and the cells must be treated to take up the dye; for examplethrough the use of a electric pulse. Alternatively, the dye canpassively enter the cells, but once taken up, it is modified such thatit cannot diffuse out of the cells. For example, enzymatic modificationof the inclusion dye may render it charged, and thus unable to diffuseout of the cells. For example, the Molecular Probes CellTracker™ dyesare fluorescent chloromethyl derivatives that freely diffuse into cells,and then glutathione S-transferase-mediated reaction produces membraneimpermeant dyes.

Suitable inclusion dyes include, but are not limited to, the MolecularProbes line of CellTracker™ dyes, including, but not limited toCellTracker™ Blue, CellTracker™ Yellow-Green, CellTracker™ Green,CellTracker™ Orange, PKH26 (Sigma), and others known in the art; see theMolecular Probes Handbook, supra; chapter 15 in particular.

In general, inclusion dyes are provided to the cells at a concentrationranging from about 100 ng/ml to about 5 μg/ml, with from about 500 ng/mlto about 1 μg/ml being preferred. A wash stop may or may not be used. Ina preferred embodiment, a candidate bioactive agent is combined with thecells as described herein. The cells and the inclusion dye are incubatedfor some period of time, to allow cell division and thus dye dilution.The length of time will depend on the cell cycle time for the particularcells; in general, at least about 2 cell divisions are preferred, withat least about 3 being particularly preferred and at least about 4 beingespecially preferred. The cells are then sorted as outlined below, tocreate populations of cells that are replicating and those that are not.As will be appreciated by those in the art, in some cases, for examplewhen screening for anti-proliferation agents, the bright (i.e.fluorescent) cells are collected; in other embodiments, for example forscreening for proliferation agents, the low fluorescence cells arecollected. Alterations are determined by measuring the fluorescence ateither different time points or in different cell populations, andcomparing the determinations to one another or to standards.

In a preferred embodiment, the proliferation assay is an antimetaboliteassay. In general, antimetabolite assays find the most use when agentsthat cause cellular arrest in G1 or G2 resting phase is desired. In anantimetabolite proliferation assay, the use of a toxic antimetabolitethat will kill dividing cells will result in survival of only thosecells that are not dividing. Suitable antimetabolites include, but arenot limited to, standard chemotherapeutic agents such as methotrexate,cisplatin, taxol, hydroxyurea, nucleotide analogs such as AraG, etc. Inaddition, antimetabolite assays may include the use of genes that causecell death upon expression.

The concentration at which the antimetabolite is added will depend onthe toxicity of the particular antimetabolite, and will be determined asis known in the art. The antimetabolite is added and the cells aregenerally incubated for some period of time; again, the exact period oftime will depend on the characteristics and identity of theantimetabolite as well as the cell cycle time of the particular cellpopulation. Generally, a time sufficient for at least one cell divisionto occur.

In a preferred embodiment, at least one proliferation assay is run, withmore than one being preferred. Thus, a proliferation assay results in apopulation of proliferating cells and a population of arrested cells.Moreover, other proliferation assays may be used, i.e., colorimetricassays known in the art.

In a preferred embodiment, either after or simultaneously with one ormore of the proliferation assays outlined above, at least one cell phaseassay is done. A “cell phase” assay determines at which cell phase thecells are arrested, M, G1, S, or G2.

In a preferred embodiment, the cell phase assay is a DNA binding dyeassay. Briefly, a DNA binding dye is introduced to the cells, and takenup passively. Once inside the cell, the DNA binding dye binds to DNA,generally by intercalation, although in some cases, the dyes can beeither major or minor groove binding compounds. The amount of dye isthus directly correlated to the amount of DNA in the cell, which variesby cell phase; G2 and M phase cells have twice the DNA content of G1phase cells, and S phase cells have an intermediate amount; depending onat what point in S phase the cells are. Suitable DNA binding dyes arepermeant, and include, but are not limited to, Hoechst 33342 and 33258,acridine orange, 7-AAD, LDS 751, DAPI, and SYTO 16, Molecular ProbesHandbook, supra; chapters 8 and 16 in particular.

In general, the DNA binding dyes are added in concentrations rangingfrom about 1 μg/ml to about 5 μg/ml. The dyes are added to the cells andallowed to incubate for some period of time; the length of time willdepend in part on the dye chosen. In one embodiment, measurements aretaken immediately after addition of the dye. The cells are then sortedas outlined below, to create populations of cells that contain differentamounts of dye, and thus different amounts of DNA; in this way, cellsthat are replicating are separated from those that are not. As will beappreciated by those in the art, in some cases, for example whenscreening for anti-proliferation agents, cells with the leastfluorescence (and thus a single copy of the genome) can be separatedfrom those that are replicating and thus contain more than a singlegenome of DNA. Alterations are determined by measuring the fluorescenceat either different time points or in different cell populations, andcomparing the determinations to one another or to standards.

In a preferred embodiment, the cell phase assay is a cyclin destructionassay. In this embodiment, prior to screening (and generally prior tothe introduction of a candidate bioactive agent, as outlined below), afusion nucleic acid is introduced to the cells. The fusion nucleic acidcomprises nucleic acid encoding a cyclin destruction box and a nucleicacid encoding a detectable molecule. “Cyclin destruction boxes” areknown in the art and are sequences that cause destruction via theubiquitination pathway of proteins containing the boxes duringparticular cell phases. That is, for example, G1 cyclins may be stableduring G1 phase but degraded during S phase due to the presence of a G1cyclin destruction box. Thus, by linking a cyclin destruction box to adetectable molecule, for example green fluorescent protein, the presenceor absence of the detectable molecule can serve to identify the cellphase of the cell population. In a preferred embodiment, multiple boxesare used, preferably each with a different fluor, such that detection ofthe cell phase can occur.

A number of cyclin destruction boxes are known in the art, for example,cyclin A has a destruction box comprising the sequence RTVLGVIGD (SEQ IDNO:48): the destruction box of cyclin B1 comprises the sequenceRTALGDIGN (SEQ ID NO:51). See Glotzer et al., Nature 349:132-138 (1991).Other destruction boxes are known as well: YMTVSIIDRFMQDSCVPKKMLQLVGVT(SEQ ID NO:36; rat cyclin B); KFRLLQETMYMTVSIIDRFMQNSCVPKK (SEQ IDNO:37; mouse cyclin B); RAILIDWLIQVQMKFRLLQETMYMTVS (SEQ ID NO:38; mousecyclin B1); DRFLQAQLVCRKKLQVVGITALLLASK (SEQ ID NO:39; mouse cyclin B2);and MSVLRGKLQLVGTAAMLL (SEQ ID NO:40; mouse cyclin A2).

The nucleic acid encoding the cyclin destruction box is operably linkedto nucleic acid encoding a detectable molecule. The fusion proteins areconstructed by methods known in the art. For example, the nucleic acidsencoding the destruction box is ligated to a nucleic acid encoding adetectable molecule. By “detectable molecule” herein is meant a moleculethat allows a cell or compound comprising the detectable molecule to bedistinguished from one that does not contain it, i.e., an epitope,sometimes called an antigen TAG, a specific enzyme, or a fluorescentmolecule. Preferred fluorescent molecules include but are not limited togreen fluorescent protein (GFP), blue fluorescent protein (BFP), yellowfluorescent protein (YFP), red fluorescent protein (RFP), and enzymesincluding luciferase and β-galactosidase. When antigen TAGs are used,preferred embodiments utilize cell surface antigens. The epitope ispreferably any detectable peptide which is not generally found on thecytoplasmic membrane, although in some instances, if the epitope is onenormally found on the cells, increases may be detected, although this isgenerally not preferred. Similarly, enzymatic detectable molecules mayalso be used; for example, an enzyme that generates a novel orchromogenic product.

Accordingly, the results of sorting after cell phase assays generallyresult in at least two populations of cells that are in different cellphases.

The proteins and nucleic acids provided herein can also be used forscreening purposes wherein the protein-protein interactions of the BTRXhor NTR proteins can be identified. Genetic systems have been describedto detect protein-protein interactions. The first work was done in yeastsystems, namely the “yeast two-hybrid” system. The basic system requiresa protein-protein interaction in order to turn on transcription of areporter gene. Subsequent work was done in mammalian cells. See Fieldset al., Nature 340:245 (1989); Vasavada et al., PNAS USA 88:10686(1991); Fearon et al., PNAS USA 89:7958 (1992); Dang et al. Mol. Cell.Biol. 11:954 (1991); Chien et al., PNAS USA 88:9578 (1991); and U.S.Pat. Nos. 5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463, apreferred system is described in Ser. Nos. 09/050,863, filed Mar. 30,1998 and 09/359,081 filed Jul. 22, 1999, entitled “Mammalian ProteinInteraction Cloning System”. For use in conjunction with these systems,a particularly useful shuttle vector is described in Ser. No.09/133,944, filed Aug. 14, 1998, entitled “Shuttle Vectors”.

In general, two nucleic acids are transformed into a cell, where one isa “bait” such as the gene encoding an BTRXh or NTR protein or a portionthereof, and the other encodes a test candidate. Only if the twoexpression products bind to one another will an indicator, such as afluorescent protein, be expressed. Expression of the indicator indicateswhen a test candidate binds to the BTRXh or NTR protein and can beidentified as an BTRXh or NTR protein. Using the same system and theidentified BTRXh or NTR proteins the reverse can be performed. Namely,the BTRXh or NTR proteins provided herein can be used to identify newbaits, or agents which interact with BTRXh or NTR proteins.Additionally, the two-hybrid system can be used wherein a test candidateis added in addition to the bait and the BTRXh or NTR protein encodingnucleic acids to determine agents which interfere with the bait and theBTRXh or NTR protein interactions.

In one embodiment, a mammalian two-hybrid system is preferred. Mammaliansystems provide post-translational modifications of proteins which maycontribute significantly to their ability to interact. In addition, amammalian two-hybrid system can be used in a wide variety of mammaliancell types to mimic the regulation, induction, processing, etc. ofspecific proteins within a particular cell type. For example, proteinsinvolved in a disease state (i.e., cancer, apotosis related disorders)could be tested in the relevant disease cells. Similarly, for testing ofrandom proteins, assaying them under the relevant cellular conditionswill give the highest positive results. Furthermore, the mammalian cellscan be tested under a variety of experimental conditions that may affectintracellular protein-protein interactions, such as in the presence ofhormones, drugs, growth factors and cytokines, radiation,chemotherapeutics, cellular and chemical stimuli, etc., that maycontribute to conditions which can effect protein-protein interactions,particularly those involved in cancer.

Assays involving binding such as the two-hybrid system may take intoaccount non-specific binding proteins (NSB).

Expression in various cell types, and assays for BTRXh or NTR activityare described above. The activity assays, such as having an effect on,for example, the oxidation/reduction state of a cell or cell component,organelle, or molecule performed to confirm the activity of BTRXh or NTRproteins which have already been identified by their sequenceidentity/similarity or binding to BTRXh or NTR as well as to furtherconfirm the activity of lead compounds identified as modulators of BTRXhor NTR.

In one embodiment, the BTRXh or NTR proteins of the present inventionmay be used to generate polyclonal and monoclonal antibodies to BTRXh orNTR proteins, which are useful as described herein. Similarly, the BTRXhor NTR proteins can be coupled, using standard technology, to affinitychromatography columns. These columns may then be used to purify BTRXhor NTR antibodies. In a preferred embodiment, the antibodies aregenerated to epitopes unique to the NTR protein; that is, the antibodiesshow little or no cross-reactivity to other proteins. These antibodiesfind use in a number of applications. For example, the BTRXh or NTRantibodies may be coupled to standard affinity chromatography columnsand used to purify BTRXh or NTR proteins as further described below. Theantibodies may also be used as blocking polypeptides, as outlined above,since they will specifically bind to the BTRXh or NTR protein.

The anti-NTR protein antibodies may comprise polyclonal antibodies.Methods of preparing polyclonal antibodies are known to the skilledartisan. Polyclonal antibodies can be raised in a mammal, for example,by one or more injections of an immunizing agent and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. The immunizing agent may include the BTRXh or NTR protein ora fusion protein thereof. It may be useful to conjugate the immunizingagent to a protein known to be immunogenic in the mammal beingimmunized. Examples of such immunogenic proteins include but are notlimited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. Examples of adjuvantswhich may be employed include Freund's complete adjuvant and MPL-TDMadjuvant (monophosphoryl Lipid a, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

The anti-Btrxh or ant-NTR protein antibodies may, alternatively, bemonoclonal antibodies. Monoclonal antibodies may be prepared usinghybridoma methods, such as those described by Kohler and Milstein,Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, orother appropriate host animal, is typically immunized with an immunizingagent to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the immunizing agent.Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include the BTRXh or NTR protein ora fusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred in immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against NTRprotein. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunosorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteina-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,587. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

The anti-NTR protein antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science. 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe NTR protein, the other one is for any other antigen, and preferablyfor a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

The anti-NTR protein antibodies of the invention have various utilities.For example, anti-NTR protein antibodies may be used in diagnosticassays for a NTR protein, e.g., detecting its expression in specificcells or tissues etc. Various diagnostic assay techniques known in theart may be used, such as competitive binding assays, direct or indirectsandwich assays and immunoprecipitation assays conducted in eitherheterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: aManual of Techniques, CRC Press, Inc. (1987) pp. 147-158]. Theantibodies used in the diagnostic assays can be labeled with adetectable signal. The detectable moiety should be capable of producing,either directly or indirectly, a detectable signal. For example, thedetectable moiety may be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or¹²⁵I, a fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase. Any methodknown in the art for conjugating the antibody to the detectable moietymay be employed, including those methods described by Hunter et al.,Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Painet al., J. Immunol. Meth., 40:219 (1981): and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-Btrxh or anti-NTR protein antibodies also are useful for theaffinity purification of BTRXh or NTR protein from recombinant cellculture or natural sources. In this process, the antibodies against NTRprotein are immobilized on a suitable support such a Sephadex resin orfilter paper, using methods well known in the art. The immobilizedantibody then is contacted with a sample containing the BTRXh or NTRprotein to be purified, and thereafter the support is washed with asuitable solvent that will remove substantially all the material in thesample except the BTRXh or NTR protein, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent that will release the BTRXh or NTR protein from theantibody.

III. Use of Plants Expressing Elevated Levels of Thioredoxin and/or NTR

In one embodiment, the transgene protein, for example BTRXh or NTRtransgene expressed in plants (see for example U.S. Ser. No.60/126,736), especially seeds or grains, using the methods describedherein, is used in the production and synthesis of BTRXh or NTR. TheBTRXh or NTR transgene expressed by the recombinant nucleic acid of theinvention may be harvested at any point after expression of the proteinhas commenced. When harvesting from the seed or grain or other part of aplant for example, it is not necessary for the seed or grain or otherpart of the plant to have undergone maturation prior to harvesting. Forexample, transgene expression may occur prior to seed or grainmaturation or may reach optimal levels prior to seed or grainmaturation. The transgene protein may be isolated from the seeds orgrain, if desired, by conventional protein purification methods. Forexample, the seed or grain can be milled, then extracted with an aqueousor organic extraction medium, followed by purification of the extractedthioredoxin protein. Alternatively, depending on the nature of theintended use, the transgene protein may be partially purified, or theseed or grain may be used directly without purification of the transgeneprotein for food processing or other purposes.

The overexpression of the BTRXh or NTR either alone or, preferably incombination, in a seed of grain increases the redox status (SH:SS ratio)of the seed or grain. The combination can be achieved by, for example,breeding plants individually transformed with either BTRXh or NTR,co-transformation with BTRXh and NTR expression vectors, or by mixingthe products of the individually transformed plants. In a preferredembodiment, the transgenic seed or grains of the invention find use inthe production of food or feed products with increased digestibility,decreased allergenicity, a redistribution of the protein of a seed orgrain to the more soluble faction.

For example, the addition of thioredoxin promotes the formation of aprotein network that produces flour with enhanced baking quality.Kobrehel et al., (1994) have shown that the addition of thioredoxin toflour of non-glutenous cereal such as rice, maize, and sorghum promotesthe formation of a dough-like product. Accordingly, the addition ofthioredoxin expressed in seeds using the methods described herein finduse in the production of flour with improved baking quality such asincreased strength and/or volume.

The enhanced expression of thioredoxin also produces a seed having analtered biochemical composition. For example, enhanced thioredoxinexpression produces seed with increased enzymatic activity, such as,increased pullulanase and alpha-amylase A. Enhanced thioredoxinexpression also produces seed with early alpha-amylase B activation.Pullulanase (“debranching enzyme”) is an enzyme that breaks downbranched starch of the endosperm of cereal seeds by hydroliticallycleaving alpha-1,6 bonds. Alpha-amylases break down starch 1-4 linkages.Pullulanase and amylases are enzymes fundamental to the brewing andbaking industries. Pullulanase and amylases are required to break downstarch in malting and in certain baking procedures carried out in theabsence of added sugars or other carbohydrates. Obtaining adequateactivity of these enzymes is problematic especially in the maltingindustry. It has been known for some time that dithiothreitol (DTT, achemical reductant that reduces and sometimes replaces thioredoxin)activates pullulanase of cereal preparations (e.g., barley, oat, andrice flours). A method of adequately increasing the activity ofpullulanase and alpha-amylase A and shortening the activation time ofalpha-amylase B with a physiologically acceptable system, leads to morerapid malting methods and, owing to increased sugar availability, toalcoholic beverages such as beers with reduced carbohydrate content.

Accordingly, seeds or grains with enhanced thioredoxin expressionprovide advantages in the production of malt and beverages produced by afermentation process. Enhanced pullulanase and alpha-amylase A andearlier induction of alpha-amylase B in grain increases the speed andefficiency of germination, important in malting, where malt is producedhaving increased enzymatic activity resulting in enhanced hydrolysis ofstarch to fermentable carbohydrates, thereby, improving the efficiencyof fermentation in the production of alcoholic beverages, for example,beer and scotch whiskey. Early alpha-amylase B activation would reducethe total time for malting by about 20%. Enhanced fermentation processesalso find use in the production of alcohols that are not intended forhuman consumption, i.e., industrial alcohols.

In another embodiment, seed or grains with enhanced thioredoxinexpression provide advantages in enhancing the onset and efficiency ofgermination.

The overexpression of thioredoxin in seed or grains results in anincrease in the total protein. It also promotes the redistribution ofproteins to the most soluble albumin/globulin fraction and theproduction of flour and other food products, feed, and beverages withimproved digestibility in comparison to edible products made fromnon-transformed grains. Such edible products find use in ameliorationand treatment of food malabsorptive syndromes, for example, sprue orcatarrhal dysentery. Sprue is a malabsorptive syndrome affecting bothchildren and adults, precipitated by the ingestion of gluten-containingfoods. Edible products that are more readily digested and readilyabsorbed avoid or ameliorate the disease symptoms. Edible products withimproved digestibility also ameliorate or reduce symptoms associatedwith celiac disease in which storage proteins that are not readilydigested in afflicted individuals result in inflammation of the GItract.

The expression of thioredoxin in seed grains results in the productionof foods and other edible products with reduced allergenicity incomparison to edible products made from non-transformed grains. Foodallergies are a significant health and nutrition problem (Lehrer et al.,1996). Up to 2% of adults and 8% of children have a food allergy causingserious symptoms including death. Wheat protein is one of the principalallergens. Food allergies are defined by the American academy of Allergyand Immunology Committee on Adverse Reactions to Food as “animmunological reaction resulting from the ingestion of a food or a foodadditive” (Fenema, 1996; Lasztity, 1996). Most true allergic responsesto food proteins appear to be caused by a type-I immunoglobulin E(IgE)-mediated hypersensitivity reaction (Sicherer, 1999). Theseresponses may occur within minutes or a few hours after eating theoffending food (Furlong-Munoz, 1996). When the offending food isingested by allergy-sensitive individuals the body releases histaminesand other biochemicals, resulting in itchy eyes, rash or hives; runnynose; swelling of the lips, tongue, and face; itching or tightness ofthe throat; abdominal pain; nausea; diarrhea; and shortness of breath.Some individuals have severe, anaphylactic reactions, resulting inapproximately 135 deaths per year in the United States. In the U.S. over2,500 emergency rooms visits per year are allergy-related. There is nocure for food allergies, only avoidance of the food will preventsymptoms. For example, patients with wheat allergy must avoid wheat- orgluten-containing foods; wheat gluten is a very common ingredient inmany processed foods (Marx et al., 2000).

A feature common to many allergens is the presence of one or moredisulfide bonds that contribute to the resistance of allergens todigestion (Astwood et al., 1996), allowing them to be mostly intact whenthey react the small intestine where they are presented to mucosal cellsthat mount an IgE immune response. The major allergens were found to beinsoluble storage proteins, gliadins and glutenins. The soluble storageproteins, albumins and globulins were considerably weaker (Buchanan etal., 1997). Allergenicity of these proteins is substantially deceasedafter thioredoxin treatment and disulfide bond reduction.

Edible products, for example, bread, cookies, dough, thickeners,beverages, malt, pasta, food additives, including animal feeds, madeusing the transgenic plants or parts of a transgenic plant of theinvention have decreased allergenicity and accordingly can be used to inthe treatment of an allergic response. By “treatment” or “alleviating”symptoms herein is meant prevention or decreasing the probability ofsymptoms.

Increased digestibility of seeds or grains also provides widerconsumption of grains by man and animals who otherwise can not consumesuch grains. For example, sorghum is the world's fifth leading grain interms of metric tons after wheat, rice, maize, and barley and third inproduction in the Untied States after maize and wheat. The use ofsorghum is constrained in part because of the difficulty associated withthe digestibility of its protein and starch compared to other grains.This difficulty with the digestibility of sorghum protein and starch hasto do with the structure of the seed and the manner in which theproteins are associated with the starch. The digestibility of the starchflour from sorghum cultivars is 15-25% lower in digestibility than, forexample, maize. Perhaps more notable is the fact that, unlike othergrains, the indigestibility of unprocessed sorghum flour increasesdramatically after boiling in water, a common practice in Africa. Astudy with human subjects showed that protein digestibility in cookedsorghum porridge can be as low as 46%, whereas the percent digestibilityfor cooked wheat, maize, and rice was 81%, 73%, and 66% respectively(Mertz et al. 1984, MacLean et al. 1981). Exogenous addition of reducingagents increases the digestibility of the starch (Hamaker et al. 1987).However, the efficacy of manipulating the thioredoxin system in vivo inthe seed by expressing increased amounts of thioredoxin in a mannerwhich does not adversely affect plant development or morphology had notpreviously been demonstrated. Accordingly, the transgenic plants of theinvention provide wider use of seeds or grains as food sources byincreasing the digestibility of the starch and/or protein component. Thetransgenic seeds or grains of the present invention also provide theadvantage of increasing the digestibility of food products for human andfeed for animals made of these grains without the addition of exogenousreducing agents. In addition, the increased digestibility results ingreater utilization of the food or feed, i.e., a human or animalconsuming an edible product comprising a transgenic seed or grain of theinvention or an extract thereof more efficiently absorbs nutrients andtherefore requires to consume less in comparison to a non-transgenicfood product. In another embodiment the transgenic seed, grain orextracts thereof of the present invention and extracts or food productsthereof are used as a food or feed additives. For example, an extract orflour or malt produced from a transgenic seed or grain of the inventionis added to a non-transgenic food or feed product to improve thedigestibility or decrease the allergenicity of the nontransgenic foodproduct or to improve the quality of the total food product, such as, byincreasing the strength and/or volume of the food product.

Illustrative embodiments of the invention are described below.

EXAMPLES Example 1 Barley Gene for Thioredoxin h

Barley thioredoxin h was cloned using PCR with primers derived from theknown sequences of two thioredoxin h wheat genes (Gautier et al., 1998).When these two sequences were compared, conserved amino acid regionswere found. The following primers were prepared that hybridized to theseregions:

wtrh4: 5′-CCAAGAAGTTCCCAGCGTC-3′ (SEQ ID NO:7) wtrh2R5′-CACGCGGCGGCCCAGTAA-3′. (SEQ ID NO:8)

These primers were used in an amplification reaction essentially asdescribed by Sambrook et al. (1989). A scutellum barley (Hordeum vulgareL.) cDNA library was used as template. The resultant PCR product,corresponding to part of the barley thioredoxin h sequence highlyhomologous to the wheat cDNAs (FIG. 2), was gel-purified using QIAquickGel extraction kit (Quiagen, UK) and sequenced using an automatedsequencer (Perken Elmer, CA, USA). This amplification product was thenused to build a gene-specific probe according to a random primingprotocol (Promega, Madison, Wis., USA) using ³²P-dCTP. The synthesizedprobe was purified with a TE Midi Select-D, 650 column (5 Prime-3 Prime,Inc., CO, USA), and used to screen the barley scutellum λZapII cDNAlibrary. Plaques were transferred onto nitrocellulose filters(NitroPure, MSI, Westboro, Mass., USA) by standard methods (Sambrook etal., 1989). The DNA was fixed onto the filters using a Stratalinker UVcrosslinking apparatus (Stratagene, La Jolla, Calif., USA) andprehybridized for 3.5 hours at 55° C. in a MKII Mini Oven (Hybaid,Woodridge, N.J., USA) using a solution containing 6×SSC, 10 mM EDTA,5×Denhardt's solution, 0.5% SDS and 100 ug/ml of boiled calf thymus DNA(Sambrook et al., 1989). Hybridization was carried out at 68° C. for 15hours with 30 ul of the barley thioredoxin h probe-solution perhybridization. Blots were washed twice in 2×SSC, 0.1% SDS at roomtemperature for 30 minutes, twice in 1×SSC, 0.1% SDS at 65° C. for 30minutes, and once in 0.1×SSC, 0.1% SDS, then exposed to X-ray film at−70° C. with two intensifying screens for 18 hours.

Hybridizing plaques were isolated separately using a Pasteur pipette andresuspended in 500 μl SM (Sambrook et al., 1989) with 20 μl chloroformin an Eppendorf tube. The samples were then vortexed for two minutes andstored at 4° C. overnight. The phage suspension was diluted so thatapproximately 100 plaques were contained on each 100 mm plate (one coredplaque in 1 ml SM buffer represents about 0.10⁵ pfu (plaque formingunit(s); Lambda ZapII Library Instruction Manual, Stratagene, La Jolla,Calif.). Two positive clones per 20,000 plaque-forming units (pfu) werefound. After a second screening purification, the size of the insert inthe positive clones was determined using the T3 and T7 primers whichhybridize to the extremities of the λ ZapII polylinker site. Two 1.5 kbfragments were obtained. Sequencing revealed that these two clonescontained the same full-length thioredoxin h cDNA.

The full-length barley thioredoxin h cDNA is 369 bp (FIG. 2) and encodesa protein of MW 13,165 Daltons (FIG. 1) with a theoretical pi of 5.12.It shares homology with the Arabidopsis and wheat thioredoxin h cDNAs,but is unique in its nucleic and amino acid sequences. The putativecorresponding amino acid sequence contains the conserved thioredoxinactive site (FIG. 1). The barley amino acid and cDNA sequences arehomologous to known wheat thioredoxins h sequences (FIGS. 1 and 2).Nevertheless, the alanine enriched amino-terminal region is shorter inthe barley thioredoxin h (by 5 amino acids compared to pTaM1338(accession number X69915) and by 8 amino acids compared to pTd14132(accession number AJ001903) (FIG. 1)).

Example 2 Nucleotide Sequence of a cDNA Encoding an NADP-ThioredoxinReductase (NTR) from Barley (Hordeum vulgare L.)

A cDNA library from barley (Hordeum vulgare L., cv. Himalaya) scutellumtissues was constructed by in λ Zap II (Stratagene, La Jolla, Calif.)from poly(A)+ RNA. The cDNA library was screened by PCR using a set ofdegenerate primers, mRNTR2 (5′-TTCTTCGCSATCGGMCAYGARCC-3′; SEQ ID NO:13)plus mRNTR5R (5′-GCGTCSARRGCRGCCATGCASCC-3′; SEQ ID NO:14), producingthe internal 201-bp fragment. It was rescreened using a set of primers,MPNTR7 (5′-ACSACSACSACSGACGTSGARAA-3′; SEQ ID NO:15) plus BNTR9R(5′-ACTGGTATGTGTAGAGCCC-3′; SEQ ID NO:16), producing the internal 693-bpfragment (FIG. 3). The sequences of 5′- and 3′-cDNA ends with cDNAlibrary were obtained by PCR using primer sets, T3(5′-AATTAACCCTCACTAAAGGG-3′; SEQ ID NO:17) plus BNTR12R(5′-AAGTTCTCGACGTCGGTGGTG-3′; SEQ ID NO:18) and M13R(5′-CAGGAAACAGCTATGAC-3′; SEQ ID NO:19) plus BNTR10(5′-ATTATGCAGGCTAGGGCGCTC-3′; SEQ ID NO:20), respectively. A full-lengthbarley scutellum NTR cDNA clone was amplified by PCR using a primer set,BNTR22 (5′-TATCTAGAATGGAGGGATCCGCCGCGGCGC-3′; SEQ ID NO:21) plus BNTR23R(5′-TTGGTACCTCAATCAGACTTGCCCACCTGT-3′; SEQ ID NO:22), was subcloned intothe pAct1INosKmf(−) vector at the XbaI-KpnI sites and the PCR-amplifiedNTR sequence was then identified by DNA sequencing analysis.

The barley scutellum NTR cDNA clone has an open reading frame (ORF) of332 amino acids (SEQ ID NO:23) (FIGS. 5A-B; Table 1). The calculatedmolecular weight determined for the translation product of that ORF was34,900 daltons and the predicted PI is 6.03 (Table 1). The barleyscutellum deduced amino acid sequence has 71% similarity with the A.thaliana NTR (SEQ ID NO:24) and 39% with E. coli NTR (SEQ ID NO:25)(FIG. 4B) using the CLUSTAL-V method set at default parameters (Higginsand Sharp, (1989) Comput. Appl. Biosci., 5(2):151-153). A gene treeanalysis suggested that the sequence of H. vulgare NTR is more closelyrelated to that of A. thaliana NTR than E. coli NTR (FIG. 4C).

FIGS. 5A and 5B show the nucleotide sequence of the barley scutellum NTR(SEQ ID NO:10) isolated from a cDNA library. At the nucleotide level, H.vulgare NTR shows 58% similarity to A. thaliana NTR (SEQ ID NO:26) and41% to E. coli NTR (SEQ ID NO:27) (FIG. 5C) as determined by CLUSTAL-Vdefault parameters. Shaded residues in FIG. 5B indicate nucleotidesequences conserved in all three NTR genes from H. vulgare, A. thalianaand E. coli.

TABLE 1 Predicted Structural Class of the Whole Protein: Alpha Deléage &Roux Modification of Nishikawa and Ooi (1987) Analysis Whole ProteinMolecular Weight 34899.50 m.w. Length 332 1 microgram = 28.654 pMolesMolar Extinction Coefficient 27910 ± 5% 1 A(280) 1.25 mg/ml IsoelectricPoint   6.03 Charge ay pH 7 −4.05 Whole Protein Composition Analysis:Amino Acid(s) Number Count % by Weight % by Frequency Charged 81 31.0724.40 (R, K, H, Y, C, D, E) Acidic (D, E) 33 11.53 9.94 Basic (K, R) 2811.57 8.43 Polar 80 25.05 24.10 (N, C, Q, S, T, Y) Hydrophobic 127 36.6738.25 (A, I, L, F, W, V) A Ala 44 8.96 13.25 C Cys 5 1.48 1.51 D Asp 175.61 5.12 E Glu 16 5.92 4.82 F Phe 14 5.90 4.22 G Gly 37 6.05 11.14 HHis 7 2.75 2.11 I Ile 17 5.51 5.12 K Lys 12 4.41 3.61 L Leu 19 6.16 5.72M Met 7 2.63 2.11 N Asn 11 3.60 3.31 P Pro 12 3.34 3.61 Q Gln 11 4.043.31 R Arg 16 7.16 4.82 S Ser 21 5.24 6.33 T Thr 24 6.95 7.23 V Val 308.52 9.04 W Trp 3 1.60 0.90 Y Tyr 8 3.74 2.41 B Asx 0 0.00 0.00 Z Glx 10.37 0.30 X Xxx 0 0.00 0.00 Ter 0 0.00 0.00

Example 3 Expression of Wheat Thioredoxin h (WTRXh) in Transgenic Barley

Four different DNA constructs were produced, each containing a 384-bpwtrxh fragment encoding the 13.5-KDa WTRXh protein. The four constructsare illustrated in FIG. 6 and described below. Each construct comprisedthe 384-bp wtrxh fragment operably linked to a seed-specific promoter(either the barley endosperm-specific D-hordein or B1-hordein promotersor the maize embryo-specific globulin promoter). An additional constructcomprised the 384-bp wtrxh fragment operably linked to the B1-hordeinpromoter and the B1-hordein signal sequence (FIG. 6). The transformationvector used included the bar gene, conferring resistance to bialaphos.Twenty-eight independent regenerable barley lines were obtained afterbialaphos selection and all were PCR-positive for the bar gene. Thepresence of the wtrxh gene was confirmed in the genome of the 28independent lines by PCR and DNA hybridization analyses. The expressionof the WTRXh protein was assessed by western blot analysis, usingpurified wheat thioredoxin as a control. The WTRXh expressed intransgenic barley had a molecular mass that differed from native barleyTRXh but was identical to WTRXh. The WTRXh was found to be highlyexpressed in developing and mature seed of transgenic barley plantsalthough levels of expression varied among the transgenic events. Onaverage, higher expression levels were observed in lines transformedwith the DNA construct containing the B1-hordein promoter plus thesignal peptide sequence than the same promoter without the signalpeptide sequence. The WTRXh purified from transgenic barley seed wasconfirmed to be biochemically active.

A. Materials and Methods

Plant Materials for Transformation

A two-rowed spring cultivar of barley, Golden Promise, was grown ingrowth chambers as described previously (Wan and Lemaux 1994; Lemaux etal., 1996).

Construction of Wheat Thioredoxin h Expression Vectors and DNASequencing

Expression vectors were constructed containing the wheat thioredoxin hgene (wtrxh) driven by the barley endosperm-specific B1- or D-hordeinpromoter or the maize embryo-specific globulin promoter. The plasmidswere constructed as follows.

(1) pDhWTRXN-2: A 384-bp wtrxh coding region was amplified by PCR frompTaM13.38 (Gautier et al., 1998). This plasmid contained a cDNA ofwtrxh, which was used as a template, creating XbaI and SacI sites withthe following primers Wtrxh1 (5′-atatctagaATGGCGGCGTCGGCGGCGA) (SEQ IDNO:28) and Wtrxh2R (5′-atagagctcTTACTGGGCCGCGTGTAG) (SEQ ID NO:29),respectively (FIG. 6). Small letters in the primer denote a restrictionenzyme site for subcloning of the DNA fragment containing the wtrxhgene; underlined letters denote wtrxh sequences. The ATG initiationcodon for wtrxh expression was included in the Wtrxh1 primer. PCRreactions were performed on a thermocycler (MJ Research Inc., Watertown,Mass.) using recombinant Taq DNA polymerase (Promega, Madison, Wis.) ina 100-μl reaction volume. The reaction buffer contained 10 mM Tris-HCl(pH 9.0), 50 mM KCl, 1.5 mM MgCl₂, 0.1% Triton-X-100, and 50 μM of eachdeoxyribonucleoside triphosphate. PCR conditions utilized 25 cycles of94° C. for 1 min, 55° C. for 1 min and 72° C. for 2 min, with a finalextension step at 72° C. for 7 min. The wtrxh fragment, which wasamplified with the primers Wtrxh1 and Wtrxh2R, was purified from a 0.7%agarose gel using a QIAquick® gel extraction kit (Qiagen Inc.,Chatsworth, Calif.), digested with XbaI and SacI and ligated intoXbaI/SacI-digested pUC19 to generate the pWTRXh-1 plasmid. Nucleotidesequences of the PCR-amplified wtrxh coding region fragment weredetermined by the dideoxynucleotide chain termination method usingSequenase according to manufacturer's instructions (United StatesBiochemical, Cleveland, Ohio) with double-stranded plasmid templates andregularly spaced primers.

pDhWTRXN-2 was made by replacing the uidA gene in pDhGN-2 (containingbarley endosperm-specific D-hordein promoter (FIG. 12) and nos 3′terminator) with the XbaI/SacI fragment containing the wtrxh codingsequence from pWTRXh-1, which contains the PCR-amplified wtrxh codingsequence in pUC19. To construct pDhGN-2, a 0.4-kb D-hordein promoter wasamplified by PCR from pDII-Hor3 (Sørenson et al., 1989, 1996; Cho etal., 1999a). This plasmid contained the D-hordein promoter sequence,which was used as a template, creating SphI and XbaI sites with thefollowing primers: Dhor1 (5′-ggcgcatgcgaattcGAATTCGATATCGATCTTCGA-3′)(SEQ ID NO:30) and Dhor2 (5′-aactctagaCTCGGTGGACTGTCAATG-3′) (SEQ IDNO:31), respectively. Small letters in the primers contain restrictionenzyme sites for subcloning of the DNA fragment containing the D-hordeinpromoter; underlined letters denote D-hordein promoter sequences. ThePCR amplified D-hordein promoter fragment was digested with SphI andXbaI and replaced with the cauliflower mosaic 35S (CaMV 35S) promoter inp35SGN-3 to generate the pDhGN-2 plasmid. p35SGN-3 was made by ligatingthe 3.0-kb SphI-EcoRI fragment containing the CaMV 35S promoter, uidA(beta-glucuronidase, gus) gene and nos into the SphI/EcoRI-digestedpUC18.

(2) pdBhWTRX-1: The construction of pdBhWTRXN-1 started by usingpBhWTRXN-1, pBhWTRXN-1 was made by replacing the uidA gene in pBhGN-1,which contains uidA driven by the barley endosperm-specific B1-hordeinpromoter and terminated by the nos 3′ terminator, with the XbaI/SacIfragment from pWTRXh-1, which contains the wtrxh coding sequence. The120-bp HindIII-5′ B1-hordein flanking region was deleted from thepBhWTRXN-1 and religated to make the pdBhWTRXN-1 construct.

(3) pdBhssWTRXN3-8: Primers Bhor7 (5′-GTAAAGCITTAACAACCCACACATTG) (SEQID NO:7) and BhorWtrxh1R (5′-CCGACGCCGCTGCAATCGTACTTGTTGCCGCAAT) (SEQ IDNO:8) containing HindIII and AcyI sites, respectively, were used foramplification of a 0.49-kb B1-hordein 5′-region, which included theB1-hordein signal peptide sequence (FIG. 11). A λ2-4/HindIII plasmidcontaining a genomic clone of B1-hordein (Brandt et al., 1985; Cho andLemaux, 1997) was used as a template for the amplification. The primerBhorWtrxh1R is an overlapping primer, which contains the wtrxh codingsequence (underlined) and a partial signal peptide sequence from theB1-hordein promoter, but lacks the ATG initiation codon for wtrxh.pdBhssWTRXN3-8 was made by replacing the D-hordein promoter (FIG. 6) inpDhWTRXN-2 with the 0.49-kb PCR-amplified HindIII/AcyI fragment, whichcontains the B1-hordein promoter, its signal peptide sequence and thejunction region from the 5′ trxh gene. Thus, construct pdBhssWTRXN3-8contains the barley endosperm-specific B1-hordein promoter with itssignal peptide sequence (FIG. 6), wtrxh, and nos (FIG. 6). The signalpeptide sequence containing the ATG initiation codon was directlycombined with the sequence of wtrxh, with no extra amino acid sequencesbeing introduced between the two. This ensures that the WTRXh proteinhas a precise cleavage site in the lumen of the endoplasmic reticulum(ER). The authenticity of a PCR-amplified fragment from the chimericproduct was confirmed by DNA sequencing.

(4) pGIb1WTRXN-1: The 1.42-kb HindIII/BamHI fragment containing themaize embryo-specific globulin promoter from the ppGIb1GUS plasmid (Liuand Kriz, 1996) was ligated into pBluescript II KS(+) to create HindIIIand XbaI sites. pGIbWTRXN-1 was made by restricting pDhWTRXN-2 withHindIII and XbaI in order to remove the 0.49-kb HindIII/XbaI barleyD-hordein promoter from the pDhWTRXN-2. In place of the 0.49-kbHindIII/XbaI D-hordein promoter fragment (FIG. 6), the 1.42-kbHindIII/XbaI maize globulin promoter was ligated into the HindIII/XbaIdigested pDhWTRXN-2 to form the pGIbWTRXN-1 plasmid.

Stable Barley Transformation

Stable transgenic lines of barley expressing WTRXh driven by theB1-hordein promoter with and without the signal peptide sequence (FIG.11 SEQ ID NO:11), by the D-hordein promoter (FIG. 12 SEQ ID NO:12) andby the maize globulin promoter were obtained following modifications ofpublished protocols (Wan and Lemaux 1994; Lemaux et al., 1996; Cho etal., 1998a-c). Whole immature embryos (IEs) (1.0-2.5 mm) wereaseptically removed, placed scutellum-side down on DC callus-inductionmedium containing 2.5 mg/L 2,4D and 5 μM CuSO₄ (Cho et al., 1998a-c).One day after incubation at 24±1° C. in the dark, the IEs weretransferred scutellum-side up to DC medium containing equimolar amountsof mannitol and sorbitol to give a final concentration of 0.4 M. Fourhours after treatment with the osmoticum, the IEs were used forbombardment. Gold particles (1.0 μm) were coated with 25 μg of a 1:1molar ratio of pAHC20 (Christensen and Quail, 1996) and one of thefollowing plasmids, pdBhWTRXN-1, pdBhssWTRXN3-8, pDhWTRXN-2 andpG1bWTRXN-1. The microprojectiles were bombarded using a PDS-1000 Hebiolistic device (Bio-Rad, Inc., Hercules, Calif.) at 1100 psi.Bombarded IEs were selected on DC medium with 5 mg/L bialaphos for 2 to3 months. Bialaphos-resistant callus was transferred onto anintermediate culturing medium (DBC2; Cho et al., 1998a-c), containing2.5 mg/L 2,4D, 0.1 mg/L BAP and 5.0 μM CuSO4, between the selection [DCmedium plus bialaphos (Meiji Seika Kaisha, Ltd., Yokohama, Japan)] andregeneration (FHG medium; Hunter, 1988) steps. The culturing aftercallus induction and selection on DC medium were carried out under dimlight conditions (approximately 10 to 30 μE, 16 h-light) (Cho et al.,1998a-c). Regenerated shoots were transferred to Magenta boxescontaining rooting medium (callus-induction medium withoutphytohormones) containing 3 mg/L bialaphos. When shoots reached the topof the box, plantlets were transferred to sod in the greenhouse.

Cytological Analysis

For cytological analysis of transgenic barley plants, healthy rootmeristems were collected from young plants grown in the greenhouse.After pretreatment at 4° C. in saturated 1-bromonaphthalene solutionovernight, root meristems were fixed in 1:3 glacial acetic acid:ethanoland stored at 4° C. Root meristems were hydrolyzed in 1 M HCl at 60° C.for 5-7 min, stained in Feulgen solution and squashed on a glass slidein a drop of 1% aceto-carmine. Chomosomes were counted from at leastfive well-spread cells per plant.

Herbicide Application

To determine herbicide sensitivity of T₀ plants and their progeny, asection of leaf blade at the 4- to 5-leaf stage was painted using acotton swab with 0.25% (v/v) Basta™ solution (starting concentration 200g/L phophinothricin, Hoechst AG, Frankfurt, Germany) plus 0.1% Tween 20.Plants were scored 1 week after herbicide application.

Polymerase Chain Reaction (PCR) and DNA Blot Hybridation

Total genomic DNA from leaf tissues was purified as described byDellaporta (1993). To test for the presence of wtrxh in genomic DNA ofputatively transformed lines, 250 ng of genomic DNA was amplified by PCRusing one of two primer sets:

Set 1: (SEQ ID NO:28) Wtrxh1 (5′-ATATCTAGAATGGCGGCGTCGGCGGCGA) and (SEQID NO:29) Wtrxh2R (5′-ATAGAGCTCTTACTGGGCCGCGTGTAG); or Set 2: (SEQ IDNO:32) Wtrxh4 (5′-CCAAGAAGTTCCCAGCTGC) and (SEQ ID NO:33) Wtrxh5R(5′-ATAGCTGCGACAACCCTGTCCTT). The presence of bar was determined usingthe primer set: (SEQ ID NO:34) BAR5F (5′-CATCGAGACAAGCACGGTCAACTTC3′)and (SEQ ID NO:35) BAR1R (5′-ATATCCGAGCGCCTCGTGCATGCG) (Lemaux et al.,1996).

Amplifications were performed with Taq DNA polymerase (Promega, Madison,Wis.) in a 25-μl reaction (Cho et al., 1998a-c). Twenty-five microlitersof the PCR product with loading dye were subjected to electrophoresis ina 1.0% agarose gel with ethidium bromide and photographed using exposureto UV light. Presence of 0.4- and 0.14-kb fragments was consistent withintact and truncated wtrxh fragments, respectively; an internal 0.34-kbfragment was produced from the bar gene with bar primers. Homozygouslines for wtrxh were screened by PCR and western blot analysis in T₂ orT₃ plants.

For DNA hybridization analysis, 10 μg of total genomic DNA from leaftissue of each line was digested with HindIII and SacI, separated on a1.0% agarose gel, transferred to Zeta-Probe GT membrane (Bio-Rad,Hercules, Calif.) and hybridized with a radiolabeled wtrxh-specificprobe following the manufacturer's instructions. The wtrxh-containing0.4 kb XbaI-SacI fragment from pDhWTRXN-9 was purified by QIAEX gelexaction kit (QIAGEN, Chatsworth, Calif.) and labeled with ³²P-dCTPusing random primers.

Western Blot Analysis

Western blot analysis was performed on seeds from selected transgeniclines as well as from control barley seeds from non-transgenic GoldenPromise grown under the same conditions as the transgenic plants andfrom control wheat seeds of a durum wheat cultivar, cv. Monroe, or abread wheat cultivar cv. Capitale. Whole seeds were ground to a finepowder with a mortar and pestle under liquid nitrogen. Ten to 20 seedswere used for each sample; the volume of extraction buffer (50 mM TrisHCl or phosphate buffer, pH 7.8, 0.5 mM phenylmethyl sulfonyl fluoride[PMSF], 1 mM EDTA) varied from 2 to 4 ml depending on the number ofseeds used and the viscosity of the extract. Grinding was continued foran additional minute after buffer addition; the mixture was thencentrifuged at 14,000×g for 10 minutes and the supernatant solution wassaved as the album-globulin fraction that contained the thioredoxin.

SDS-PAGE of the albumin-globulin fraction was performed in 12-17%polyacrylamide gradient gels at pH 8.5 (Laemmli, 1970). From each sampleequal amounts of protein (˜40 μg) quantitated according to Bradford(1976) were diluted 1:2 v/v in Laemmli sample buffer, boiled for 3minutes, loaded onto gels and subjected to electrophoresis at a constantcurrent of 15 mA. Proteins were transferred to nitrocellulose at aconstant voltage of 40 V for 4 hours at 4° C. using a Hoefer TransphorTransfer Unit (Alameda, Calif.). Nitrocellulose was blocked with 5%powdered milk in TBS for 2 hours at room temperature (RT), incubated inprimary antibody for 4 hours at RT and in secondary antibody for 1 hourat RT. Primary antibody was wheat anti-thioredoxin h II Ab (Johnson etal., 1987b) diluted 1 to 500; secondary antibody was goat anti-rabbitalkaline phosphatase (Bio-Rad, Hercules, Calif.) diluted 1:3000. Blotswere developed in NBT/BCIP alkaline phosphatase color reagent (accordingto Bio-Rad instructions); gels were stained with Coomassie blue toassure transfer. Images were scanned using a Bio-Rad GelDoc 1000(Hercules, Calif.) and analyzed using Bio-Rad Multi Analyst, version1.0.2. All bands were scanned over the same area, using a rectangle ofcomparable density as background; results were expressed as % of volumescanned. The number shown represents the percent of the total volume(pixel density×area of scanned band).

WTRXh Activity Measurements

Preparation of Materials for Extraction.

Mature grains from various heterozygous and homozygous transgenic linesserved as starting materials for the assay. Heterazygous lines with aD-hordein promoter were: GPDhBarWtrx-5, GPDhBarWtrx-9-1, andGPDhBarWtrx-9-2. Heterozygous lines with a B-hordein promoter and nosignal sequence were: GPdBhBarWtrx-2, -5, -9, -19 and GPdBhBarWtrx-20.Heterozygous lines with a B-hordein promoter plus a signal sequencewere: GPdBhssBarWtrx-2, -7, GPdBhssBarWtrx-29, GPdBhssBarWtrx-20,GPdBhssBarWtrx-14, GPdBhssBarWtrx-22. Homozygous lines with a signalsequence were: GPdBhssBarWtrx-2-17, GPdBhssBarWtrx-2-17-1,GPdBhssBarWtrx-29-3 and GPdBhssBarWtrx-29-3-2. Control materialsincluded a non-transformed tissue culture derived line, 4-96, atransformed line containing only bar, GPBar-1, and null segregant lines,GPdBhssBarWtrx-29-11 and GPdBhssBarWtrx-29-11-10, derived from lineGPdBhssBarWtrx-29.

Preparation of (NH₄)₂SO₄ Extracts for Gel Filtration

Approximately fifteen grams of barley grains were ground to powder in acoffee grinder and extracted with 80 ml (1:4 w/v) of buffer [(50 mMTris-HCl buffer, pH 7.9, 1 mM EDTA, 0.5 mM PMSF (phenylmethysulfonylfluoride)], 2 mM e-amino-n caproic acid, 2 mM benzamidine-HCl) bystirring for 3 hrs at 4° C. The slurry plus the rinse was subjected tocentrifugation at 25,400×g for 20 min, the supernatant solution wasdecanted through glass wool, pellets were resuspended in a small volumeof buffer and then clarified by centrifugation as before. Thesupernatant fractions were combined, an aliquot was removed and theremainder was subjected to acidificaton by adjusting the pH form 7.83 to4.80 with 2 N formic acid; denatured proteins were removed bycentrifugation as above prior to assay. The pH of the acidifiedsupernatant solution was readjusted to 7.91 with 2 N NH₄OH and analiquot was removed for assay. Powdered (NH₄₎ ₂SO₄ was added to a finalconcentration of 30% and the sample was stirred for 20 min at 4° C.,followed by centrifugation as described above. The pellet was discarded.Additional (NH₄)₂SO₄ was added to bring the decanted supernatantsolution to 90% saturation; the sample was stirred for 16 hrs at 4° C.,followed by centrifugation as described above.

The supernatant solution was discarded, the 30-90% (NH₄)₂SO₄ pelletswere resuspended in 30 mM Tris-HCl, pH 7.9 buffer and then subjected tocentrifugation at 40,00×g for 15 min to clarify. The resultingsupernatant (30-90% (NH₄)₂SO₄ fraction) was added to dialysis tubing(6,000-8,000 MW cut-off) and exposed to solid sucrose at 4° C. to obtaina 10-fold reduction in volume. An aliquot (1 ml) of the clarified andconcentrated 30-90% (NH₄)₂SO₄) sample was saved and the remaining samplewas applied to a pre-equilibrated (30 mM Tris-HCl, pH 7.9, 200 mM NaCl)Sephadex G-50 superfine column (2.5×90 cm: ˜400 mL bed volume) with aperistaltic pump at a flow rate of 0.5 mL/min. Protein was eluted withthe same buffer at the same flow rate; one hundred fifty drop-fractionswere collected. Selected fractions were used to measure absorbance at280 nm using a Pharmacia Biotech Ultrospec 4000 and to assay for TRXhactivity following the NADP-MDH activation protocol (see below). Activefractions were pooled, stored at 4° C., and then assayed for totalNADP-MDH activation activity.

Preparation of Heat-Treated Extracts

Approximately 10 grams of barley grains were ground to powder for about30 sec in a coffee grinder and extracted by shaking for 1 hr at roomtemperature in 60 mL buffer as above. The slurry plus the rinse wassubjected to centrifugation at 27,000×g for 20 min and the supernatantsolution decanted through glass wool. A 20 mL aliquot of each sample washeated at 65° C. until sample temperature reached 60±1° C. (˜10 min).The sample was held at 60° C. for 10 additional min, followed by coolingin an ice/water bath. The cooled sample was centrifuged and thesupernatant solution was concentrated by sucrose as above and stored at−20° C. Frozen samples were thawed and clarified by centrifugation at14,000 rpm for 10 min at 4° C. Total TRXh activity was estimated on theconcentrated, supernatant fractions.

NADP-Malate Dehydrogenase Activation Assay

Thioredoxin h activity was assayed as previously described (Florencio etal. 1988; Johnson et al., 1987a). Fifty to 120 μl of extract (dependingon activity) was preincubated with DTT, and 0.16 to 0.32 μl of thepre-incubation mixture was used for the NADP-MDH assay. Control assayswere conducted on identical fractions in the absence of NADP-MDH.Western blot analysis was conducted as described above except that 10 to20% SDS-polyacrylamide gels were used for electrophoresis and transferto nitrocellulose paper was for 4 hrs at 40 V.

Sequential Extraction of Multiple Protein Fractions

Ten grams of barley grain were sequentially extracted for albumin(H₂O-soluble), globulin (salt-soluble), hordeins (alcohol-soluble) andglutelins (Shewry et al., 1980). Barley powder was stirred with 0.5 MNaCl for 1 h at 25° C. to remove salt-soluble proteins. Two sequentialhordein fractions were extracted from the residue with 50% propanol inthe absence (hordein-I) and presence (hordein-II) of 2% (v/v)2-mercaptoethanol. Glutelins were extracted from the residue with 0.05 Mborate buffer, pH 10, containing 1% (v/v) 2-mercaptoethanol and 1% (v/v)sodium dodecylsulphate.

In vitro Monobromobimane (mBBr) Labeling of Proteins

Immature, mature, or germinating seeds from nontransformed andtransgenic plants were ground in 100 mM Tris-HCl buffer, pH 7.9.Reactions were carried out following the protocol of Kobrehel et al.,(1992). Seventy microliters of the buffer mixture containing a knownamount of protein was either untreated or treated with DTT to a finalconcentration of 0.5 mM. After incubation for 20 min, 100 nmol of mBBrwas added, and the reaction was continued for another 15 min. To stopthe reaction and derivatize excess mBBr, 10 μl of 10% SDS and 100 μl of100 mM 2-mercaptoethanol were added. The samples were applied to a 15%SDS-PAGE gel. Fluorescence of mBBr was visualized by placing gels on alight box fitted with a UV light source (365 nm). Protein determinationwas carried out by the Bradford dye binding method (Bradford 1976) usingbovine serum albumin or gamma globulin as standards.

Assay of Pullulanase and its Inhibitor

To measure pullulanase activity, grain was germinated in a dark chamberand retained for up to 5 days at 25° C. as described (Kobrehel et al.,1992.; Lozano et al., 1996). A set of plates from each line was removedfor extract preparation each day. Cell-free endosperm extracts wereprepared from lots of 10-20 germinated grains of equivalent root andcoleoptile length within a given cohort. Endosperm was separated fromthe embryo and other tissues and added to Tris-HCl buffer (50 mM, pH7.9) supplemented with 1 mM EDTA and 0.5 mM PMSF (1:3 to 1:6, wt/volratio of tissue to buffer depending on developmental stage). Aftergrinding in a mortar on ice, the sample was clarified by centrifugation(10 min at 24,000×g); the supernatant fraction was recovered and storedin 0.5-ml aliquots −80° C. for pullulanase spectrophotometric or gelassays.

Pullulanase activity was determined spectrophotometerically at 37° C. bymeasuring dye released after 30 min at 534 nm using red pullulan(Megazyme, Bray, Ireland) as substrate in 50 mM citrate-phosphate buffer(pH 5.2) (Serre et al., 1990.). Pullulanase also was assayed on nativeactivity gels of 7.5% acrylamide, 1.5 mm thickness, containing 1% redpullulan (Furegon et al., 1994.). Gels were scanned using a Bio-Rad GelDoc 1000 and analyzed using Bio-Rad MULTI ANALYST, version 1.0.2.Pullulanase inhibitor activity was determined on fractions heated toinactivate pullulanase (70° C. for 15 min) by measuring their ability toinhibit added purified barley malt pullulanase. Endogenous pullulanaseactivity was shown to be completely eliminated by this heat-treatmentwhile the inhibitor activity was not affected (Macri et al., 1993;MacGregor et al., 1994).

Alpha-Amylase Activity in Barley Grain Overexpressing Thioredoxin h

Amylase activity from the null segregant and homozygous barley grainswas analyzed during germination and early seedling growth by using gelscontaining starch. Native polyacrylamide electrophoresis gels [6%acrylamide, 1.5 mm thick] were prepared and developed according to themethod of Laemmli (1970) except that SDS was omitted from all solutions.The separating gel contained 0.5% soluble starch (Lintner potato starch,Sigma Chemical Co., St Louis, Mo.). Lyophilized samples were dissolvedin distilled H₂O and mixed 1:1 with a buffer consisting of 0.25 MTris-HCl, pH 6.8, 50% glycerol, 0.04% bromophenol blue, and 3 mM CaCl₂.Fifty micrograms of sample protein were loaded in each lane.Electrophoresis was carried out at 80 milliamps per gel at 4° C. untilthe dye front was at the edge of the gel (usually 4 to 5 hours). Afterelectrophoresis, the gels were incubated in 100 ml of 0.1 M succinatebuffer, pH 6.0, for 1-2 hours at 37° C. The gels were then stained for 5min in a solution containing 2.5 mM I₂ and 0.5 M KI. Gels were washed indistilled H₂O. Except for the white regions containing amylase activity,gels were stained dark blue.

Isoelectricfocusing (IEF)

For determination of alpha-amylase isozyme patterns, extracts from bothdry and germinating grain of transformed and control (untransformed)barley were separated by electophoresis at 4° C. [1.0 mm thick, pH 3-10isoelectric focusing (IEF) polyacrylamide gels, using the X cell IIsystem (NOVEX, San Diego, Calif.)]. Cathode buffer contained 20 mMarginine, and 20 mM lysine; anode buffer was 7 mM phosphoric acid.Samples were mixed 1:1 and 2×IEF sample buffer pH 3-10 (NOVEX). Aftersample application (20 μg/lane) gels were developed at constant voltage[100 V for 1 hr, 200 V for an additional 1 hr, and 500 V for 30 min].IEF standards (Bio-Rad) were used to determine the pH gradient of thegels.

Multiple Antibody Probing of IEF Gels

Western blot analysis of alpha-amylase isozymes was performed using aMini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). Seed extractsfrom the null segregant and homozygous lines overexpressing wheatthioredoxin h were separated by IEF gels as described above. Proteinswere transferred to nitrocellulose at a constant voltage of 100 V for 1hr at 4° C. using 0.75% acetic acid as blotting buffer. Nitrocellulosewas blocked with 5% powdered milk in Tris buffer solution (20 mMTri-HCl, pH 7.5, supplemented with 0.15 M NaCl) for 1 hr at roomtemperature, incubated with primary antibody for 4 hours at roomtemperature and then with secondary antibody for 1 hour at roomtemperature. Primary antibody was anti-barley alpha-amylase B diluted1:1000; secondary antibody was goat anti-rabbit alkaline phosphatase(Bio-Rad) diluted 1:3000. Blots were developed in NBT/BCIP alkalinephosphatase color reagent (according to Bio-Rad instructions) therebyrendering the cross-reacted alpha-amylase bluish-purple. To achieve fullidentity of isozyme pattern, blots were probed a second time withanother primary antibody, anti-alpha-amylase A (diluted 1:1000) and thesecondary antibody (as above). This time blots were developed inNaphthol Phosphate/Fast Red alkaline phosphatase color reagent(according to Bio-Rad instructions) which gave a pink stain to thealpha-amylase A. The blot shown was subject to this dual probingprecedure.

B. Results and Discussion

Production of Transgenic Plants

One day after bombardment, the whole embryos were transferred onto DCmedium with 5 mg/L bialaphos. At transfer to the second selection plate(5 mg/L bialaphos), all material from individual callusing embryos wasbroken into small pieces (2-4 mm) using forceps and maintainedseparately. During the subsequent two to five selection passages on 5mg/L bialaphos (at 10-20 d intervals). callus pieces showing evidence ofmore vigorous growth were transferred to new selection plates. Duringthe second round of selection, some pieces of callus were inhibited ingrowth and in some cases pieces turned brown. In general, transformedtissues were observed after three or more rounds of selection. Thebialaphos-resistant tissues were transferred onto an intermediatemedium, DBC2 or DBC3 (Cho et al., 1998a-c) with bialaphos (5 mg/L), andgrown for 1 to 2 months before regeneration on FHG medium containing 3mg/L bialaphos. Green plantlets were transferred into Magenta boxescontaining 3 mg/L bialaphos. Twenty-eight independent putativelytransformed, regenerable lines were produced after bialaphos selection(shown in Table 3).

TABLE 3 Transgenic Barley Lines Transformed with Wheat Thioredoxin hGene. DNA PCR Plasmids for Transgenic (T₀ leaf) TRXh ExpressionBombardment Barley Line bar wtrxh in T₁ seeds Ploidy CommentspdBhWTRXN-1 + GPdBhBarWTRX-1 + + n.d. Tetraploid pAHC20GPdBhBarWTRX-2 + + + Tetraploid GPdBhBarWTRX-3 + + + DiploidGPdBhBarWTRX-5 + + + Tetraploid Sterile GPdBhBarWTRX-16 + − n.d.Tetraploid GPdBhBarWTRX-17 + + n.d. Tetraploid GPdBhBarWTRX-19 + + +Diploid GPdBhBarWTRX-20 + + + Diploid GPdBhBarWTRX-22 + + + DiploidGPdBhBarWTRX-23 + + + Diploid pdBhssWTRXN3-8 + GPdBhssBarWTRX-1 + − −Diploid pAHC20 GPdBhssBarWTRX-2 + + + Diploid HomozygousGPdBhssBarWTRX-3 + + − Diploid GPdBhssBarWTRX-7 + + + DiploidGPdBhssBarWTRX-9 + + n.d. Tetraploid GPdBhssBarWTRX-11 + + − DiploidGPdBhssBarWTRX-13 + + + Tetraploid GPdBhssBarWTRX-14 + + + DiploidGPdBhssBarWTRX-20 + + + Tetraploid GPdBhssBarWTRX-21 + + n.d. TetraploidSterile GPdBhssBarWTRX-22 + + + Tetraploid GPdBhssBarWTRX-29 + + +Diploid Homozygous pDhWTRXN-2 + GPDhBarWTRX-5 + + + Tetraploid pAHC20GPDhBarWTRX-7 + + + Diploid GPDhBarWTRX-8 + + + DiploidGPDBhBarWTRX-9 + + + Diploid Homozygous GPDBhBarWTRX-22 + + + DiploidSterile pGlbWTRXN-1 + GPGlbBarWTRX-1 + + + Diploid pAHC20 *n.d.: notdetermined

Analysis of T₀ Plants and their Progeny

PCR analysis was performed using two sets of WTRXh primers and one setof BAR primers (see FIG. 6). PCR amplification resulted in 0.4-kb intactwtrxh or 0.14 kb truncated wtrxh and 0.34-kb internal bar fragments fromtransgenic lines. Of the 28 lines tested, 28 yielded bar fragments fromT₀ leaf tissue and 26 produced PCR-amplified fragments for wtrxh, givinga 93% co-transformation frequency. Nine lines were transformed withpdBhWTRXN-1, eleven with pdBhssWTRXN-8, five with pDhWTRXN-2 and onewith pG1bWTRXN-1 (see Table 2). Three lines (GPdBhBarWtrx-5,GPdBhssBarWtrx-21 and GPDhBarWtrx-22) were sterile. Seeds of T₁ plantsand their progeny from selected wtrxh-positive lines were planted inorder to screen for homozygous lines. Homozygous lines and nullsegregants were obtained from GPdBhssBarWtrx-2, -29 and GPDhBarWtrx-9(see Table 2).

Cytological Analysis of Transgenic Plants

Chromosomes were counted in root meristem cells of independentlytransformed T₀ barley plants. Out of 28 independent transgenic linesexamined. 17 lines had the normal diploid chromosome complement(2n=2×=14), while the remaining 11 lines were tetraploid (2n=4×=28) (seeTable 2 ).

Characterization and Content of WTRXh Produced in Transgenic Seed

As discussed above, several stably transformed barley lines wereobtained that express wheat thioredoxin h. As seen in FIG. 7, the stableintroduction of the wtrxh linked to the B1-hordein promoter with thesignal peptide sequence resulted in greatly enhanced expression ofactive WTRXh in transgenic barley seed.

Analysis by western blot of soluble protein fractions of the three linesin which the thioredoxin gene was linked to a signal sequence(GPdBhssBarWtrx-22, GPdBhssBarWtrx-29 and GPdBhssBarWtrx-7) showeddifferences in the level of expression (shown in Table 3). LineGPdBhssBarWtrx-22, GPdBhssBarWtrx-29 and GPdBhssBarWtrx-7, respectively,showed 22 times, 10 times and 5.5 times more WTRXh protein thannontransformed control seeds. The analyses showed that the thioredoxincontent of the null segregant (GPdBhssBarWtrx-29-11) was approximatelyhalf that of the corresponding control. The three lines generated fromthe construct in which the thioredoxin gene was not associated with asignal sequence were also compared to nontransformed control barley seedand they exhibited the following increases in TRXh levels as indicatedby the western blot analyses: GPDhBarWtrx-9; 12 times; GPDhBarWtrx-5:6.3 times; GPdBhBarWtrx-2: 6.4 times. When probed on Western Blots, thetransgenic lines show two bands while the control barley generaly showsonly one and in some cases a second minor band. Furthermore, the tissuesfrom the transgenic lines were characterized by a band that did notcorrespond to either of the barley bands but did correspond to wheatthioredoxin h. These data indicate that the protein introduced bytransformation is wheat thioredoxin h.

TABLE 3 Western Blot Analyses of Overexpression of Wheat Thioredoxin hin Barley. Fold Increase Barley Line % Volume Scanned (or Decrease)Non-Transformed Control: Golden Promise 1.46 1.0 Transformed with SignalSequence: GPdBhssBarWtrx-22 32.44 22 GpdBhssBarWtrx-29 14.62 10GpdBhssBarWtrx-7 7.99 5.5 Transformed without Signal Sequence:GPDhBarWtrx-9 17.69 12 GPDhBarWtrx-5 9.20 6.3 GPdBhBarWtrx-2 9.29 6.4Null Segregant: GPdBhssBarWtrx-29-11-10 0.93 (0.64)

The Wheat Thioredoxin h in Barley Grains is Biologically Active

Because of interference from other enzymes that oxidize NADPH, theactivity of TRXh cannot be accurately assayed in crude extracts, therebynecessitating its partial purification. Partially purified extracts ofthe different transgenic and control lines were prepared from 15 gramsof seed using ammonium sulfate fractionation and gel filtrationchromatography. Activity was measured with an NADP-MDH activation assay.Profiles based on these assays show that the activity of TRXh in thetransformed seed is much higher than in the nontransformed control (seeFIG. 7.) The activity results are summarized in Table 4.

Total WTRXh activity from the seeds of two lines transformed with theB1-hordein promoter and the signal sequence (GPBhssBarWtrx-3;GPdBhssBarWtrx-29) is about 4- to 10-fold higher, respectively, thanthat of control, nontransformed seed. Total activity from a linetransformed with the D-hordein promoter without the signal sequence(BGPDhBbarWtrx-5) is only slightly higher (1.25-fold) than that of thenontransformed control (see Table 4). In the transgenics, the specificactivity of thioredoxin is generally about 0.128 A_(340 nm)/min/mgprotein or about two fold over null segregants.

TABLE 4 Summary of Total Buffer-Extracted Protein and Total ThioredoxinActivity from Active Fraction after Gel Filtration. Total Specific TotalActivity, Activity, Barley Line Protein, mg A₃₄₀/min A₃₄₀/min/mg Control(GP 4-96)  102.6 (1.00)*   7.4 (1.00)*  0.064 (1.00)* GPDhBarWtrx-5171.2 (1.67)  9.2 (1.2) 0.054 (0.8)  GpdBhssBarWtrx-29 149.1 (1.45) 72.0(9.7) 0.483 (7.5)  GpdBhssBarWtrx-3 231.3 (2.25) 27.7 (6.4) 0.794 (12.4)*Numbers in brackets are fold increase over that of the control.

The transformed barley grains analyzed so far appear to have more totalbuffer-extracted protein than control, nontransformed seed (Table 4).

The transformed grains have a thioredoxin content of at least about10-15 μg thioredoxin/mg soluble protein(about 2-8 μg thioredoxin/mgtissue) or about two-fold higher than the null segregant.

Because of the tediousness of the (NH₄)₂SO₄ procedure and therequirement for large quantities of seed, the original extractionprocedure was modified to include a heat treatment step. This change wasbased on the fact that E. coli WTRXh is stable after treatment at 60° C.for 10 min (Mark and Richardson, 1976). Results on WTRX from twodifferent transgenic barley seeds (GPdBhBarWtrx-3, GPdBhssBarWtr-29)showed no significant difference in activity between the heat treatedand non-heat treated extracts (FIG. 8). In addition heat-treatmentdecreased the endogenous, nonspecific activity in this assay, therebyincreasing the reliability of the measurements.

Ten different barley lines (transformed and nontransformed) wereextracted using the heat-treatment step and assayed with the NADP-MDHassay; the results are summarized in Table 5. In general, total WTRXhactivities in seeds from lines transformed with the B-hordein promoterand signal sequence linked to wtrxh are much higher (4- to 35-fold) thanin seeds from lines transformed with the same promoter without signalsequence linked to wtrxh or in seeds from the nontransformed control(Table 5). At this point it is not known whether all expressed wheatWTRXh in barley seeds is heat stable.

TABLE 5 Relative Total Thioredoxin Activity in Different TransgenicBarley Lines. Total Total Specific Line Designation Protein (%) Activity(%) Activity (%) Non-transgenic control GP4-96 100 100 100 Bar Gene OnlyGPBar-1 92 120 131 Without Signal Sequence GPdBhBarWtrx-1 101 192 190GPdBhBarWtrx-22 113 151 133 GPdBhBarWtrx-23 118 180 153 With SignalSequence GPdBhssBarWtrx-2 137 1650 1203 GPdBhssBarWtrx-14 122 1723 1418GPdBhssBarWtrx-20 147 440 299 GPdBhssBarWtrx-22 154 3470 2245GPdBhssBarWtrx-29 108 1316 1219 One hundred percent of (a) totalprotein, mg: (b) total activity, nmol/min; and (c) specific activity,nmol/min/mg protein of the non-transgenic control are: (a) 116.4; (b)157.38 (c) 1.52 respectively.

Of the stably transformed lines that expressed wheat thioredoxin h, onaverage, its level was found to be higher in transformants that had thesignal peptide-containing constructs than to those that did not (Table5). Western blot analysis of soluble protein fractions from heterozygousmixtures of seeds from three of the lines, GPdBhssBarWtrx-7,GPdBhssBarWtrx-29, and GPdBhssBarWtrx-22 showed 5.5 times, 22 times, and10 times more thioredoxin h, respectively, than nontransformed controlgrain (Table 3). The thioredoxin content of the null segregant(GPdBhssBarWtrx-29-11-10) was about half that of the corresponding,nontransformed control.

Extracts from barley typically showed one immunologically reactive band(identified by B in FIG. 9A, lanes 1 and 6) but in some transfers showeda second faint, faster moving band (FIG. 9B, lane 2). Tissues fromtransgenic lines overexpressing wtrxh were characterized by a band thatdid not correspond to either of the two counterparts in barley, butrather to thioredoxin h from wheat. The difference between theoverexpressed 13.5-kDa wheat and the endogenous 13.14-kDa barleythioredoxin h is particularly pronounced in the barley line transformedwith the nontargeted thioredoxin h gene (FIG. 9A, line 5 and FIG. 9B,lane 1). Repeated analyses of the various transgenic lines by SDS/PAGEled to the conclusion that the band identified in FIGS. 9A-B by Wcorresponds to the bread wheat wtrxh introduced by barley. Independentbiochemical assays with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)(Florencio et al., 1988.) confirmed the ability of barley NTR to reducewheat thioredoxin h (data not shown).

Because of their value in assessing biochemical attributes of the grain,homozygous wtrxh lines were identified and analyzed by Western blot. Thetwo lines identified as homozygous showed both enhanced expression ofthioredoxin h relative to that of their heterozygous parents andnontransformed controls. Analysis of GPdBhssBarWtrx-29-3 is shown inFIG. 10. It is noted that demonstration of the thioredoxin h present inthe nontransgenic control and null segregant grains (not apparent in theexposure shown in FIG. 9) required conditions that led to overexposureof the enriched transgenic preparations. Thioredoxin in the parentheterozygous grain was shown to be biochemically active.

Pullulanase and Pullulanase Inhibitor Activity in Barley GrainOverexpressing Thioredoxin h

Pullulanase is an amylolytic enzyme present in cereal grain, which has adisulfide inhibitor protein (Macri et al., 1993.; MacGregor et al.,1994.), the activity of which is linked to thioredoxin (Wong et al.,1995.). Thioredoxin reduced by NADPH via NTR, reduces the disulfidebonds of the inhibitor, allowing the targeted pullulanase enzyme to beactive. Because of this relationship, it was of interest to determinethe activity of pullulanase in the thioredoxin h-overexpressingtransformants.

Spectrophotometric assays (FIG. 13A) of extracts from transformed grainof a homozygous line (GPdBhssBarWtrx-29-3) overexpressing thioredoxin hshowed a 3- to 4-fold increase in pullulanase activity on the fifth dayafter initiation of germination relative to its null segregant.Confirmatory results were obtained in a separate experiment with nativeactivity gels. The increase in activity was apparent either when gelswere viewed directly (FIG. 13B) or when the activity on the gels wasassessed by scanning and integrating the clarified bands (FIG. 13C). Ahomozygous line isolated from a different, independent transformationevent (GPdBssBarWtrx-2-1-15) showed a similar response (data not shown).The transgenic plants expressed an pullulanase activity of about 1-2Absorbance units at 534 nm/30 min/mg protein, which is about two-foldhigher than null segregants.

Pullulanase inhibitor activity was determined on fractions heated toinactivate pullulanase (70° C. for 15 min) by measuring the inhibitionof the fractions on added purified barley malt pullulanase. Theendogenous pullulanase activity was shown to be completely eliminated bythis heat treatment whereas inhibitor activity was not affected (Macriet al., supra; MacGregor et al., supra). Analysis of comparable grainextracts revealed that the pullulanase inhibitor was inactive on thefourth and fifth days after water addition in both the transformant andnull segregants. These results thus demonstrate that the increase inpullulanase activity observed after the third day is not caused byenhanced inactivation of the inhibitor in the transgenic grain. It ispossible that thioredoxin acts either by increasing the de novosynthesis of pullulanase (Hardle et al., 1975.) or by lowering thebinding of the mature enzyme to the starchy endosperm. There is evidencethat some of the pullulanase of the mature endosperm is present in boundform and can be solubilized by reducing conditions (Sissons et al.,1993.; Sissons et al., 1994.).

Alpha-Amylase Activity in Barley Grain Overexpressing Thioredoxin h

Alpha-amylase, also an amylolytic enzyme that is induced by gibberellicacid like pullulanase, has long been considered key to germination. Thesynthesis of the major (B) and minor (A) forms of this enzyme are knownto be triggered by the hormone, gibberellic acid (GA). In addition,alpha-amylase activity is increased in vitro by the reductiveinactivation of its disulfide inhibitor protein by thioredoxin h (in thepresence of NADPH and NADP-thioredoxin reductase). The present resultswith transformed barley seeds show that, like pullulanase, thioredoxin hexpression alters alpha-amylase activity. In this case, the appearanceof the enzyme during germination is accelerated and its abundance andactivity are increased.

FIGS. 14A-D shows the early increase in both the abundance and activityof alpha-amylase (A+B forms) during germination and seedlingdevelopment. Based on the antibody response in western blots,alpha-amylase was first detected 3 days after the onset of germinationin the transgenic grain FIG. 14C) whereas the enzyme did not appearuntil the fourth day in the null segregant (FIG. 14A). The onset ofactivity (based on the activity gel) followed a similar pattern (FIG.14B and FIG. 14D). The mobility of the enzyme in the activity gel alsoreflected the early induction of activity in the transgenic grain (FIG.15). That much of this increase in activity seen early on was due to theB (a gibberellic acid-linked form) is supported by FIG. 16. Here, onecan also see that the level of the minor A form of the enzyme (alsogibberellic acid dependent) was increased in grain overexpressingthioredoxin h. Again, the appearance of significant levels of the major(B form) alpha-amylase enzyme was advanced by 1 day.

Germination of Barley Grains Overexpessing Thioredoxin h

All operations were carried out at 25° C. (unless otherwise specifiedbelow) under conditions described by Kobrehel et al. 1992 and Lozano etal. 1996. Grains were surface sterilized by continuous stirring in 0.25%bleach for 30 min. Bleach was removed by extensive washing withsterilized distilled water. Thirty sterlized null segregant(GPdBhssBarWtrx-29-22-10, in which the transgene was removed by crossingwith a self-polinated plant from the same line) and thirty sterilizedhomozygous (GPdBhssBarWtrx-29-3) seeds were placed in each of a seriesof plastic Petri dishes (12.5 cm diameter) fitted with three layers ofWhatman #1 filter paper moistened with 15 ml sterile distilled water.Plates were wrapped with aluminum foil and grain was germinated in adark chamber at 20° C. for up to 7 days. One plate was read at each timepoint shown in FIG. 17. Percent germination, in the first day (from thestart of incubation up to 24 hours), was determined by observing theemergence of the radicle. On the subsequent days, percent germinationrepresents seedling growth as determined by measuring the length ofcoleoptile and roots of the germinated grains.

The results, shown in FIG. 17, indicate that germination in transgenicbarley overexpressing wheat thioredoxin h is detected about 16 hoursafter the onset of incubation in about 25-30% of the seeds. In contrast,no germination in the null segregant was detected at 16 hours but isfirst detected 8 hours later, on Day 1. Therefore, in the transgenicgermination is advanced about 8 hours. However, on Day 1 germination wasdetected in approximately 70% or about twice the number of transgenicgrains in comparison to their null segregant counterparts. It isinteresting to note that the onset of germination in the transgenicsparallels the onset of the detection of alpha amylase as shown in FIG.15.

Sequential Extraction of Grain Proteins from Transgenic Barley Grains.

Isolated endosperm from 10 dry grains or seedlings (germinated asdescribed above) were ground with mortar and pestle at 4° C. with 3 mlTris-HCl buffer as indicated below. The separate mixtures of homozygousGPdBhssBarWtrx-29-3 and null segregant GPdBhssBarWtrx-29-22-10 grainswere place in a 5-ml screw-top centrifuge tube. Grains were mechanicallyshaken for 30 minutes and then centrifuged for 10 min at 24,000×g. Thesupernatant fraction (buffer-soluble) was decanted and saved foranalysis and the residue was extracted sequentially with the followingsolvents for the indicated times: [1] 0.5 M NaCl (30 min); [2] water (30min); [3] 2×50% propanol (2 hr); [4] 2×50% propanol+2% 2-mercaptoethanol(MET) (2 hr); and [5] 0.5 M borate buffer, pH 10, containing 1% SDS and2% 2-mercaptoethanol (2 hr). Supernatant fractions of all extracts weredetermined for volume and protein content (by Coomassie dye bindingmethod), then were stored at −20° C. until use. By convention, thefractions are designated: [1] albumin/globulin (buffer/salt/water); [2]Hordein I (propanol); [3] Hordein II (propanol+MET); and [4] glutelin(Borate/SDS/MET) (Shewry et al., 1980). These fractions were used todetermine, protein content, the distribution of proteins between thewater soluble and insoluble fractions, the total extractable protein,and reduction with NADPH.

To determine the in vivo redox status of protein from transgenic barleygrain during germination and seedling development, the extractionprocedure was repeated except that 2 mM mBBr was included in the Trisgrinding buffer and the grinding was under liquid nitrogen. The mBBrderivatized proteins were electrophoresed on SDS-polyacryamide gels (1.5mm thickness, 10-20% gels, pH 8.5 (Laemmli, 1970). Gels were developedfor 16 hr at a constant current of 8 mA. Following electophoresis, gelswere placed in 12% (w/v) trichloroacetic acid and soaked for 4 to 6 hrwith one change of solution to fix the proteins; gels were thentransferred to a solution of 40% methanol/10% acetic acid for 8 to 10 hrwith agitation to remove residual mBBr. The fluorescence of mBBr (bothfree and protein bound mBBr), was visualized by placing gels on a lightbox fitted with an ultraviolet light source (365 nm). Following removalof the excess (free) mBBr, images of gels were captured by Gel Doc 1000(Bio-Rad).

To ascertain the equivalent protein amount of loaded extracts, SDS-gelswere stained with Coomassie Brilliant Blue G-250 in 10% acetic acid for30 min, and destained in 10% acetic acid for 30 min with the aid of amicrowave oven. Protein stained gels were captured by Gel Doc 1000 asabove.

The quantification of fluorescence (pixel×mm×mm) and protein (opticaldensity×mm×mm) on gels were carried out by a software program for imageanalysis—Multi-Analyst, version 1.0 (Bio-Rad). Relative reduction wasexpressed as the ratio of fluorescence to protein.

The results of two experiments shown in Table 6, Table 7, and Table 8demonstrate an increase in the total protein on a percent grain and apercent weight basis in the transgenic barley as compared to the nullsegregant. The transgenic have a thioredoxin content that is at leasttwo-fold higher (10-15 μg/mg soluble protein; 2-8 μg/gram tissue) thanthe null segregant. The data indicate that this increase in totalextractable protein is the result in redistribution of the protein tothe most soluble albumin/globulin fraction. The redistribution of theprotein to the soluble fraction increase in the transgenics is at least5% higher than the controls.

TABLE 6 Protein Content of Various Fractions in Transgenic Barley GrainOverexpressing Wheat Thioredoxin h Experiment I* Null SegregantHomozygous Protein Fraction mg/seed mg/gram mg/seed mg/gramAlbumin/Globulin 0.482 12.25 0.546 13.58 Hordein I 0.239 6.34 0.322 8.01Hordein II 0.136 3.61 0.094 2.34 Glutelin 0.110 2.92 0.097 2.41 TotalExtractable Protein 0.947 25.12 1.059 26.34 *Weight per 10 seeds is0.377 and 0.402 full null segregant and homozygous line of transgenicbarley

TABLE 7 Protein Content of Various Fractions in Transgenic Barley GrainOverexpressing Wheat Thioredoxin h Experiment II* Null SegregantHomozygous Protein Fraction mg/seed mg/gram mg/seed mg/gramAlbumin/Globulin 0.691 20.03 1.044 27.12 Hordein I 0.373 10.81 0.36810.03 Hordein II 0.254 7.36 0.240 6.23 Glutelin 0.066 1.91 0.062 1.61Total Extractable Protein 1.384 40.11 1.732 44.99 *Weight per 10 seedsis 0.377 and 0.402 for null segregant and homozygous line of transgenicbarley

TABLE 8 Percent Increase of Extractable Protein in Homozygous Line%/grain basis %/mass basis Experiment I 12 4.9 Experiment II 25 12

Analysis of the relative redox status (SH:SS) of protein fractions intransgenic and null segregant barley grains during germination and asdry grains are shown in FIG. 18. In dry transgenic grain, the greatestincrease in reduction relative to the null segregant was observed in thehordein I fraction. This increase was paralleled by decreases in therelative redox status in the hordein II and glutelin fractions while therelative redox status of the albumin/globulin fraction was unchanged.The relative redox status of the transgenic in comparison to the nullsegregant is at least 5:1.

During germination, the albumin/globulin fraction progressivelyincreases, reaching a relative redox ratio of about 1.5 on Day 4. Therelative redox status of the hordein II and glutelin fractions alsoincreased during germination but only reached parity with the nullsegregant. In contrast the relative redox status of the hordein Ifraction was highly variable.

Example 4 Barley Thioredoxin h Gene (btrxh) Transformation Materials andMethods

Plant Material and Culture of Explants

Mature seeds of rice (Oryza saliva L. cv. Taipei 309) weresurface-sterilized for 20 min in 20% (v/v) bleach (5.25% sodiumhypochlorite) followed by 3 washes in sterile water. The seeds wereplaced on 2 different NB (Chen L et al. (1998) Plant Cell Rep 18:25-31)-based callus-induction media; (1) NBD′BC2 medium containing 2.0mg/L 2,4-D, 0.1 mg/L BAP and 0.5 μM CuSO₄ (Cho M.-J., unpublished), (2)NBDBC3 medium containing 1.0 mg/L 2,4-D, 0.5 mg/L BAP and 5.0 μM CuSO₄(Cho M.-J., unpublished). Five to 7 d after plating, germinating shootsand roots from the mature seeds were completely removed by manualexcision. After three weeks of incubation at 24+1° C. under dim-lightconditions (approximately 10 to 30 μEm⁻²s⁻¹, 16 h-light), tissues withshiny, nodular and compact structures were selected and subsequentlymaintained on NBDBC4 medium containing 0.5 mg/L 2,4-D, 2.0 mg/L BAP and5.0 μM CuSO₄ (Cho M.-J., unpublished), subculturing at 3 to 4 weekintervals, to proliferate highly regenerative, green tissues.

Construction of a Barley Thioredoxin h Expression Vector and DNASequencing

pdBhssBTRXN(Km)-2 (Cho M.-J., unpublished): the chimeric DNA constructcontaining the B₁-hordein promoter-signal sequence-btrxh (barleythioredoxin h gene) was obtained using a modified method ofsite-directed mutagenesis by PCR (Cho and Lemaux 1997). The four-primerstrategy was used to produce 2 major PCR products. Primers, Bhor7(5′-GTAAAGCTTTAACAACCCACACATTG-3′; SEQ ID NO:41) containing HindIIIrestriction site and BhorssBtrx2R(5′-CGCCGTTGCCGACGCCGCTGCAATCGTACTTGTTGCCGCAAT-3′; SEQ ID NO:42), wereused for amplification of 0.49-kb B₁-hordein 5′ region including theB₁-hordein signal peptide sequence using the λ2-4/HindIII plasmidcontaining genomic clone of B₁-hordein (Brandt et al., 1985; Cho et al.,1997) as a template. The primer BhorssBtrx2R is an overlapping primercontaining the btrxh coding sequence (italicized) and a partial signalpeptide sequence from the B₁-hordein promoter without the ATG initiationcodon for btrxh. The second PCR product was amplified using primers,BorssBtrx4 (5′-ACAAGTACGATTGCAGCGGCGTCGGCAACGGC-3′; SEQ ID NO:43) andBtrxh2R (atagagctcTTACTGGGCCGCCGCGTG; SEQ ID NO:44); cDNA donecontaining btrxh (Caillau, del Val, Cho, Lemaux and Buchanan,unpublished) was used as template. The second set of PCR reactions wasproduced 0.86-kb chimeric fragments using two PCR-amplified fragments(each diluted 50 times) and two external primers, Bhor7 and Btrx2R.

pdBhssBTRXN(Km)-2 was made by replacing the maize ubiqutin promoter inpUbilNosKmf(−) with the 0.86-kb PCR-amplified HindIII/SacI fragmentcontaining B₁I-hordein promoter with its signal peptide sequence plusbtrxh. Thus, construct pdBhssBTRXN(Km)-2 contains the barleyendosperm-specific B₁-hordein promoter with its signal peptide sequence,btrxh and nos. The signal peptide sequence containing the ATG initiationcodon was directly combined with the sequence of the btrxh gene, withouthaving extra amino acid sequences between the two, in order to makebarley thioredoxin h protein provide a precise cleavage site in thelumen of endoplasmic reticulum (ER). The PCR-amplified region of theconstruct was further confirmed by DNA sequencing, and used for stabletransformation of rice.

Stable Transformation

Approximately 4- to 5-month-old highly regenerative cultures maintainedon NBDBC4 medium were used for bombardment. Tissue pieces (3—4 mm) weretransferred for osmotic pretreatment to NBDBC4 medium containingmannitol and sorbitol (0.2 M each). Four hours after treatment withosmoticum, tissues were bombarded as previously described (Lemaux et al.1996; Cho et al. 1998). Gold particles (1.0 μm), coated with 25 μg of amixture of pAct1IHPT-4 and pdBhssBTRXN(Km)-2 at a molar ratio of 1:2were used for bombardment with a Bio-Rad PDS-1000 He biolistic device(Bio-Rad, Hercules, Calif.) at 900 or 1100 psi. Sixteen to 18 h afterbombardment, tissues were placed on osmoticum-free NBDBC4 mediumsupplemented with 20 mg/L hygromycin B and grown at 24±1° C. underdim-light (10-30 μEm⁻²s⁻¹). From the third round of selection onward,tissues were subcultured at 3- to 4-week intervals on NBDBC4 mediumcontaining 30 mg/L hygromycin B. When a sufficient amount (a plate) ofthe putatively transformed highly regenerative tissue was obtained, itwas plated on NBNBC4 medium containing 0.5 mg/L NAA, 2.0 mg/L BAP and5.0 μM CuSO₄ (Cho M.-J., unpublished) and exposed to higher intensitylight (approximately 45-55 μEm⁻²s⁻¹). Green shoots were then transferredto Magenta boxes containing phytohormone-free regeneration medium [MS(Murashige and Skoog (1962) Physiol. Plant 15:473-497) plus 20 g/Lsucrose] with 10 to 20 mg/L hygromycin B. After four weeks, regeneratedplantlets were transferred to soil.

Genomic DNA Isolation, Polymerase Chain Reaction (PCR)

Putative transgenic lines were screened by DNA PCR using two a set ofbtrxh primers, Btrxh5 (5′-CCAAGAAGTTCCCAAATGC-3′; SEQ ID NO:45) andBtrxh2R. PCR amplification resulted in 0.19-kb intact btrxh fromtransgenic lines. One btrxh-positive line (OSHptBTRX-I) was obtained.Amplifications were performed in a 25-μl reaction with Taq DNApolymerase (Promega,Madison, Wis.) as described (Cho et al. 1998).

Example 5 Barley NTR Gene (bntr) Transformation

NADP/thioredoxin system (NTS), is analogous to the system establishedfor animals and most microorganisms, in which thioredoxin (h-type inplants) is reduced by NADPH and NADP-thioredoxin reductase (NTR)(Johnson et al., 1987a, Florencio et al., 1988; Suske et al., 1979).Without being bound by theory, the NTR appears to be a limiting factorfor NTS. Therefore, we isolated barley ntr gene from barley cDNA library(Cho, Lemaux and Buchanan, unpublished) and introduce this gene intobarley, wheat, and rice plants.

Construction of a Barley NTR Expression Vector and DNA Sequencing

pActilBNTRN-4 (Cho M.-J., unpublished): pActilBNTRN-4 was made byligating the PCR-amplified XbaI/KpnI fragment containing barley ntr cDNAsequence. Primers, BNTR29 (5′attctgaATGGAGGGATCCGCCGCGGCGCCGCTC-3′; SEQID NO:46) and BNTR23R (5′-ttggtaccTCAATCAGACTTGCCCACCTGT-3′; SEQ IDNO:47), were used for amplification of the 1.012-Kb XbaI/KpnI fragmentcontaining 0.996-Kb barley ntr coding sequence; small letters contain arestriction enzyme site for subcloning of the DNA construct containingbarley ntr gene and underlined letters indicate the barley ntrsequences. The barley ntr fragment was purified from a 0.7% agarose gelusing QIAquick® gel extraction kit, digested with XbaI and KpnI andliated into XbaI/KpnI-digested pAct1lNosKmf(−) to generate thepActilBNTRN-4 plasmid. Nucleotide sequences of the PCR-amplified barleynfr coding region were determined by DNA sequencing.

Barley ntr expression vectors driven by barley endosperm-specific B₁- orD-hordein promoter with or without its signal peptide sequence areconstructed.

Stable Transformation

Transformation of barley, wheat and rice is conducted as previouslydescribed above and in Lemaux et al., 1996; Cho et al., 1998; Kim etal., 1999. Barley trxh alone, barley ntr alone or a mixture of bothgenes are used for bombardment with a Bio-Rad PDS-1000 He biolisticdevice (Bio-Rad, Hercules, Calif.) at 900 or 1100 psi. After obtainingtransgenic lines, they are analyzed for tests of redox state,germinability, allergenicity, and baking quality.

According to the above examples, other types of plants, are transformedin a similar manner to produce transgenic plants overexpressingthioredoxin and NTR either alone or in combination, such as transgenicwheat, rice, maize, oat, rye sorghum, millet, triticale, forage grass,turf grass, soybeans, lima beans, tomato, potato, soybean, cotton,tobacco etc. Further, it is understood that thioredoxins other thanwheat or barly thioredoxin or thioredoxin h can be used in the contextof the invention. Such examples include spinach h; chloroplastthioredoxin m and f, bacterial thioredoxins (e.g., E. coli) yeast, andanimal and the like. In addition, it is understood the NTR other thanbarley NTR protein also can be used in the context of the invention suchas spinach, wheat, and NTR of monocots and dicots.

This invention has been detailed both by example and by description. Itshould be apparent that one having ordinary skill in the relevant artwould be able to surmise equivalents to the invention as described inthe claims which follow but which would be within the spirit of theforegoing description. Those equivalents are included within the scopeof this invention. All herein cited patents, patent applications,publications, references and references cited therein are herebyexpressly incorporated by reference in their entirety.

REFERENCES

Ainley W M, McNeil K J, Hill J W, Lingle W L, Simpson R B, Brenner M L,Nagao R T, Key J L (1993) Regulatable endogenous production ofcytokinins up to “toxic” levels in transgenic plants and plant tissues.Plant Molecular Biology 22(1):13-23.

Altschul et al. (1990). J. Mol. Biol., 215: 403-10.

Altschul et al. (1994). Nature Genet., 6: 119-29.

Altschul et al., (1996) Methods in Enzymology, 266: 460-480.

An G, Costa M A, Mitra A, Ha SOB, Marton L (1988) Organ-specific anddevelopmental regulation of the nopaline synthase promoter in transgenictobacco plants. Plant Physiology (Rockville) 88(3):547-552.

Astwood J, Leach J N, Fuchs R L, (1996) Stability of food allergens todigestion in vitro. Nature Biotechnology, 14(10):1269-1273.

Ausubel et al. (1987) in Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley-Intersciences.

Bagga et al. (1997) Plant Cell 9:1683-1696.

Besse and Buchanan (1997) Bot. Bull. Acad. Sin. 38:1-11.

Besse I, Wong J H, Kobrehel K, Buchanan B B (1996) Thiocalsin: athioredoxin-linked, substrate-specific protease dependent on calcium.Proc Natl Acad Sci USA 93: 3169-3175

Bondenstein-Lang J, Buch A, Follman H (1989) Animal and plantmitochondria contain specific thioredoxins. FEBS Lett 258: 22-26.

Bower M S, Matias D D, Fernandes-Carvalho E, Mazzurco M, Gu T, RothsteinS J, Goring D R (1996) Two members of the thioredoxin in-family interactwith the kinase domain of a Brassica S locus receptor kinase. Plant Cell8: 1641-1650.

Bradford (1976) Anal. Biochem. 72:2 48-254.

Brandt A, Montembault A, Cameron-Mills V, Rasmussen S K (1985) Primarystructure of a B1 hordein gene from barley. Carlsberg Res Commun 50:333-345.

Bright S W, Shewry P R (1983) Improvement of protein quality in cereals.CCC Critical Plant Reviews 1: 49-93

Brugidou C, Marty I, Chartier Y, Meyer Y (1993) The Nicotiana tabacumgenome encodes two cytoplasmic thioredoxin genes which are differentlyexpressed. mol Gen Genet 238:285-293.

Buchanan B B (1991) Regulation of CO₂ assimilation in oxygenicphotosynthesis; the ferredoxin/thioredoxin system. Arch Biochem Biophys287: 337-340.

Buchanan B B, Adamidi C, Lozano R M, Yee B C, Momma M, Kobrehel K, ErmelR Frick O L (1997) Thioredoxin-linked mitigation of allergic responsesto wheat. Proc Natl Acad Sci USA 94: 5372-5377.

Buchanan et al. (1994) Arch. Biochem. Biophys. 314: 257-260.

Buchanan et al. (1998) Leatherhead Food RA Food Ind. J. 1: 97-105.

Bustos M M, Guiltinan M J, Jordano J, Begum D, Kalkan F A, Hall T C(1989) 1(9):839-853,

Callis J, Fromm M, Walbot V (1988) Heat inducible expression of achimeric maize hsp70CAT gene in maize protoplasts. Plant Physiology(Rockville) 88(4):965-968.

Cameron-Mills V (1980) The structure and composition of protein bodiespurified from barley endosperm by silica sol density gradients.Carlsberg Res Commun 45: 557-576.

Cameron-Mills V, Brandt A (1988) A B-hordein gene. Plant Mol. Biol11:449-461.

Cameron-Mills V, Madrid S M J (1988) The signal peptide cleavage site ofa B1 hordein determined by radiosequencing of the in vitro synthesizedand processed polypeptide. Carlsberg Res Commun:4:1 81-192.

Cameron-Mills V, Wettstein D von (1980) Protein body formation in thedeveloping barley endosperm. Carlsberg Res Commun 45: :77-594.

Carpenter et al. (1992) The Plant Cell 4: :557-571.

Cho M-J, Lemaux P G (1997a) Rapid PCR amplification of chimeric productsand its direct application to in vivo testing of recombinant DNAconstruction strategies. Mol. Biotechnol. 8:13-16.

Cho M-J, Vodkin I, Widholm J M (1997b) Transformation of soybeanembryogenic culture by microprojectile bombardment. Plant Biotechnol.14:11-16.

Cho M-J, Ha C D, Buchanan B B, Lemaux P G (1998a) Subcellular targetingof barley hordein promoter-uidA fusions in transgenic barley seed.P-1024. Congress in Vitro Biology, Las Vegas, Nev. 30 May-3 Jun. 1998.

Cho M-J, Jiang W, Lemaux P G (1998b) Transformation of recalcitrantcultivars through improvement of regenerability and decreased albinism.Plant Sci. 138:229-244.

Cho M-J, Zhang S, Lemaux P G (1998c). Transformation of shoot meristemtissues of oat using three different selectable markers. In Vitro CellDev. Biol. 34P:340

Cho M-J, Choi H W, Buchanan B B, Lemaux P G (1999a) Inheritance oftissue-specific expression of barley hordein promoter-uidA fusions intransgenic barley plants. Theor. Appl. Genet. 98:1253-1262.

Cho M-J, Buchanan B B, Lemaux P G (1999b) Development of transgenicsystems for monocotyledonous crop species and production of foreignproteins in transgenic barley and wheat seeds. In: Application ofTransformation Technology in Plant Breeding. Special Seminar for the30th Anniversary Korean Breeding Soc., Suwon, Korea, Nov. 19, 1999,pp.39-53.

Cho M-J, Chol H W, Lemaux P G (1999c) Transgenic orchardgrass (Dactylisglomerata L.) plants produced from high regenerative tissues. P-1089.Congress in Vitro Biology, New Orleans, La. 5-9 Jun. 1999.

Cho M-J, Jiang W, Lemaux P G (1999d) High frequency transformation ofoat via microprojectile bombardment of seed-derived regenerativecultures. Plant Sci. 148:9-17.

Cho M-J, Wong J, Marx C, Jiang W, Lemaux P G, Buchanan B B (1999e)Overexpessing of thioredoxin h leads to enhanced activity of starchdebranching enzyme (pullulanase) in germinating barley seeds. Proc.Natl. Acad. Sci. USA 96:14641-14646.

Cho M-J, Ha C D, Lemaux P G (2000) Production of transgenic tall fescueand red fescue plants by particle bombardment of mature seed-derivedhighly regenerative tissues. Plant Cell Rep. (in press).

Christensen and Quail (1996) Transgenic Res. 5: 1-6.

Conrad et al. (1998) Journal of Plant Physiology 152:708-711.

Corpet et at. (1988) Nucleic Acids Research 16: 10881-90.

del Val., Yee B C, Lozano R M, Buchanan B B, Ermel R E, Lee Y M, andFrick O L. (1999) J. Aller. Clin. Immunol. 104:690-697.

Dai S. Saarinen M, Ramaswamy S, Meyer Y, Jacquot J-P, Eklund H (1996)Crystal structure of Arabidopsis thaliana NADPH dependent thioredoxinreductase at 2.5 Å resolution J Mol Biol 264:1044-1057.

Dellaporta S (1993) Plant DNA miniprep and microprep. Freeling M, WalbotV (eds) in: Maize Handbook. p 522-525.

Dekeyser R A, Claes B, De Rycke R M U, Habets M E, Van Montagu M C,Caplan A B (1990) Transient gene expression in intact and organized ricetissues. Pant Cell 2(7):591-602.

Denis M, Delourme R, Gourret J-P, Mariani C, Renard M (1993) Expressionof engineered nuclear male sterility in Brassica napus: Genetics,morphology, cytology, and sensitivity to temperature. Plant Physiology(Rockville), 101(4):1295-1304.

Entwistle J, Knudsen S, Muller M, Cameron-Mills V (1991) Amber codonsuppression: the in vivo and in vitro analysis of two C-hordein genesfrom barley. Plant Mol Biol 17:1217-1231.

Florencio et al. (1988) Arch. Biochem. Biophys. 266: 496-507.

Forde B G, Heyworth A, Pywell J, Kreis M (1985) Nucleotide sequence of aB1 hordein gene and the identification of possible upstream regulatoryelements in endosperm storage protein genes from barley, wheat andmaize. Nucl Acids Res 13: 7327-7339.

Fromm H, Katagiri F, Chua N H (1989) An octopine synthase enhancerelement directs tissue-specific expression and binds ASF-1, a factorfrom tobacco nuclear extracts.

Furgon L, Curioni A. Peruffo A D. (1994) Anal. Biochem. 221:200-201.

Gatz C. (1997) Chemical control of gene expression. Jones R L (Ed)Annual Review of Plant Physiology and Plant Molecular Biology 48:89-108.

Gautier et al. (1998) Eur. J. Biochem. 252: 314-324.

Gelvin et al. (1990) Plant Molecular Biology Manual, Klower AcademicPublishers.

Giese H, Andersen B, Doll H (1983) Synthesis of the major storageprotein, hordein, in barley. Pulse-labeling study of grain filling inliquid-cultured detached spikes. Planta 159: 60-65

Gilmartin et al. (1992) The Plant Cell 4: 839-949.

Grimwade et al. (1996) Plant Molecular Biology 30: 1067-1073.

Hardie D G. (1975) Phytochem. 14:1719-1722.

Harlow & Lane (1988) Antbodies, A Laboratory Manual, Cold Spring HarborLaboratory, New York.

Higgins and Sharp (1988) Gene, 73: 237-244.

Higgins and Sharp (1989) CABIOS 5: 151-153.

Horecka T, Perecko D, Kutejova E, Muchova K, Kolloarova M (1996)Purification and partial characterization of two thioredoxins fromStreptomyces aureofaciens. Biochemistry and Molecular BiologyInternational 40(3):497-505.

Huang, et al. (1992) Computer Applications in the Biosciences 8: 153-65.

Hunter C P (1988) Plant regeneration from microspores of barley, Hordeumvulgare. PhD thesis. Wye College, University of London, Ashford, Kent

Innis et al. (eds.) (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc., San Diego, Calif.

Ishiwatari et al. (1995) Planta 195(3): 456-463.

Jaoquot J-P, Rivera-Madrid R. Marinho P, Kollarova M, Le Maréchal P,Miginiac-Maslow M, Meyer Y (1994) Arabidopsis thaliana NADPH thioredoxinreductase cDNA characterization and expression of the recombinantprotein in Escherichia coli. J Mol Biol 235:1357-1363.

Jiao J., Yee B C, Kobrehel K, Buchanan B B (1992). Effect ofthioredoxin-linked reduction on the activity and stability of the Kunitzand Bowman-Birk soybean trypsin inhibitor proteins. J: Agric. Food Chem40: 2333-2336.

Johnson T C, Cao R Q, Kung J E, Buchanan B B, Holmgren (1987a)Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells.Planta 171: 321-331

Johnson T C, Wada K, Buchanan B B, Holmgren A (1987b) Reduction ofpurothionin by the wheat seed thioredoxin system and potential functionas a secondary thiol messenger in redox control. Plant Physiol 85:446-431

Kim et al. (1999). P-1021 Congress on in Vitro Biol, New Orleans, La.5-9 Jun. 1999.

Kobrehel et al. (1994) Thioredoxin-linked reduction of wheat storageproteins. II. Technological Consequences. In Gluten Proteins: 1993.Association of Cereal Research; Detmold, Germany.

Kobrebel K, Wong J H, Balogh A, Kiss F, Yee B C, Buchanan B B (1992)Specific reduction of wheat storage proteins by thioredoxin h. PlantPhysiol 99: 919924

Kobrehel K. Yee B C, Buchanan B B (1991) Role of the NADP/thioredoxinsystem in the reduction of ct-amylase and trypsin inhibitor proteins. JBiol Chem 266: 16135-16140

Kuhlemeier et al. (1989) Plant Cell 1: 471.

Kuriyan J, Krishna T S R, Wong L, Guenther B, Pahler A, Williams C H,Model P (1991) Convergent evolution of similar function in 2structurally divergent enzymes. Nature 352:172-174.

Leammli, U K (1970) Cleavage of structural proteins during the assemblyof the head of bacteriophage T4. Nature 997: 680-685.

Laurent T C, Moore E C, Reichard P (1964) Enzymatic synthesis ofdeoxy-rebonucleotides IV. Isolation and characterization of thioredoxin,the hydrogen donor from Escherichia coli B. J Biol Chem 239:3436-3444.

Lemaux P G, Cho M-J, Louwerse J, Williams R, Wan Y (1996)Bombardment-mediated transformation methods for barley. Bio-Rad US/KGBulletin 2007: 1-6

Li X, Nield J, Hayman D, Langridge P (1995) Thioredoxin activity in theC terminus of Phalaris S protein. Plant J8: 133-138

Liu S. Kriz, A (1996) Tissue-specific and ABA-regulated maize Glb1 geneexpression in transgenic tobacco. Plant Cell Pep 16: 158-162

Lozano R M, Wong J H, Yee B C, Peters A, Kobrehel K, Buchanan B B (1996)New evidence for a role for thioredoxin h in germination and seedlingdevelopment. Planta 200: 100-106

Lozano R M, Yee B C, Buchanan B B (1994) Thioredoxin-linked reductiveinactivation of venom neurotoxins. Arch Biochem Biophys 309:356-362.

MacGregor A W, Marci L J, Schroeder S W, Bazin S L (1994) J. Cereal Sci.20:33-41.

Marci J L, MacGregor, A W, Schoreder, S W, Bazin, S L. (1993) J. Cereal.Sci. 18:103-106.

Marcotte et al. (1989) Plant Cell 1: 969.

Marcus F, Chamberlain S H, Chu C, Masiaez F R, Shin S, Yee B C,Buchanan, B B (1991) Plant thioredoxin h: an animal-like thioredoxinoccurring in multiple cell compartments. Arch Biochem Biophys287:195-198.

Mark and Richardson (1976) Proc. Natl. Acad. Sci. USA 73:780-784.

Marris C, Gallois P. Copley J, Kreis M (1988) The 5′ flanking region ofa barley B hordein gene controls tissue and developmental specific CATexpression in tobacco plants. Plant Mol Biol 10: 359-366

Marty I, and Meyer Y (1991) Nucleotide sequence of a complementary DNAencoding a tobacco thioredoxin. Plant Mol Biol 17: 143-148

Moore E C, Reichard P, Thelander L (1964) Enzymatic synthesis ofdeoxyribonucleotides. Purification and properties of thioredoxinreductase fom Escherichia coli B. J Biol Chem 239:3445-3453.

Muller M, Knudsen S (1993) The nitrogen response of a barley C-hordeinpromoter is controlled by positive and negative regulation of the GCN4and endosperm box. Plant J 4: 343-355.

Murashige T, Skoog F (1962) A revised medium for rapid growth andbioassays with tobacco tissue cultures. Physiol Plant 15: 473-497.

Myers and Miller (1989) CABIOS 4:11-17.

Needleman and Wunsch (1970) J. Mol. Biol. 48:443.

Opperman C H, Taylor C G, Conkling M A (1994) Root-knotnematode-directed expression of a plant root-specific gene. Science(Washington, D.C.), 263(5144):221-223.

Pai E F (1991) Variations on a theme: the family of FAD-dependentNAD(P)H-(disulphide)oxidoreductases. Curr Opin Struct Biol 1:796-803.

Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:9444.

Pearson et al. (1994) Methods in Molecular Biology 24:307-331.

Rasmussen S K, Brandt A (1986) Nucleotide sequences of cDNA clones forC-hordein polypeptides. Carlsberg Res Commun 51:371-379.

Rivera-Madrid et al. (1993) Plant Physiology 102: 324-328.

Rivera-Madrid et al. (1995) Proc. Natl. Acad. Sci. USA 92:5620-5624.

Russel M, Model P (1988) Sequence of thioredoxin reductase fromEscherichia coli. Relation to other flavoprotein disulfideoxidoreductases. J Biol Chem 263:9015-9019.

Sambrook et al. (1989) in Molecular Cloning. A Laboratory Manual, ColdSpring Harbor, New. York.

Scheibe R (1991) Redox-modulation of chloroplast enzymes. A commonprinciple for individual control. Plant Physiol 96: 1-3

Shernthaner J P, Matzke M A, Matzke A J M (1988) Endosperm-specificactivity of a zein gene promoter in transgenic tobacco plants. EMBO(European Molecular Biology Organization) Journal 7(5):1249-1256.

Serre L., Lauriere C. (1990) Analytical Biochemistry. 186(2):312-315.

Shewry P R, Field J M, Kirkman M A, Faulks A J, Miflin B J. (1980). J.Exp. Botany 31:393-407.

Shi J. and Bhattacharyya M K (1996) A novel plasma membrane-boundthioredoxin from soybean. Plant Mol Biol 32:653-662.

Sissons M J, Lance R C M, Sparrow D H B. (1993) J. Cereal Sci. 7:19-24.

Sissons M J, Lance R C M, Wallace W. (1994) Cereal Chemistry.71:520-521.

Smith and Waterman (1981) Adv. Appl. Math. 2:480.

Sørensen M B, Cameron-Mills V, Brandt A (1989) Transcriptional andpost-transcriptional regulation of gene expression in developing barleyendosperm. Mol Gen Genet 217: 195-201.

Sørensen M B, Muller M, Skerritt J, Simpson D (1996) Hordein promotermethylation and transcriptional activity in wild-type and mutant barleyendosperm. Mol Gen Genet 250:750-760.

Suske G. Wagner W, Follman H (1979) NADPH thioredoxin reductase and anew thioredoxin from wheat Z Naturforsch. C 34:214-221.

Takaiwa et al. (1995) Plant Science 111:39-49.

Torrent et al. (1997) Plant Molecular Biology 34:139-149.

U.S. application Ser. No. 60/126,736.

Vogt K, Follmann H (1986) Characterization of three differentthioredoxins in wheat. Biochem Biophys Acta 873: 415-418.

Wan and Lemaux (1994) Plant Physiol. 104: 37-48.

Weissbach & Weissbach (1989) Methods for Plant Molecular Biology,Academic Press.

Wong et al. (1993) Cereal Chem. 70(1): 113-114.

Wong J H, Jiao J A, Kobrehel K, Buchanan B. (1995) Plant Physiol. 108:67

Wu et al. (1998) Plant Journal 14:673-683.

Zhen et al. (1995) Plant Physiology 109:777-786.

All references, patents, patent applications, publications andreferences cited herein are hereby incorporated by reference in theirentirety.

51 1 369 DNA Artificial Sequence Barley Thioredoxin h cDNA 1 atggcggcgtcggcaacggc ggcggcagtg gcggcggagg tgatctcggt ccacagcctg 60 gagcagtggaccatgcagat cgaggaggcc aacaccgcca agaagctggt ggtgattgac 120 ttcactgcatcatggtgcgg accatgccgc atcatggctc cagttttcgc tgatctcgcc 180 aagaagttcccaaatgctgt tttcctcaag gtcgacgtgg atgaactgaa gcccattgct 240 gagcaattcagtgtcgaggc catgccaacg ttcctgttca tgaaggaagg agacgtcaag 300 gacagggttgtcggagctat caaggaggaa ctgaccgcca aggttgggct tcacgcggcg 360 gcccagtaa 3692 122 PRT Hordeum vulgare 2 Met Ala Ala Ser Ala Thr Ala Ala Ala Val AlaAla Glu Val Ile Ser 1 5 10 15 Val His Ser Leu Glu Gln Trp Thr Met GlnIle Glu Glu Ala Asn Thr 20 25 30 Ala Lys Lys Leu Val Val Ile Asp Phe ThrAla Ser Trp Cys Gly Pro 35 40 45 Cys Arg Ile Met Ala Pro Val Phe Ala AspLeu Ala Lys Lys Phe Pro 50 55 60 Asn Ala Val Phe Leu Lys Val Asp Val AspGlu Leu Lys Pro Ile Ala 65 70 75 80 Glu Gln Phe Ser Val Glu Ala Met ProThr Phe Leu Phe Met Lys Glu 85 90 95 Gly Asp Val Lys Asp Arg Val Val GlyAla Ile Lys Glu Glu Leu Thr 100 105 110 Ala Lys Val Gly Leu His Ala AlaAla Gln 115 120 3 382 DNA Artificial Sequence Wheat Thioredoxin h cDNA 3atggcggcgt cggcggcgac ggcgacggcg acggcggcgg cggtaggggc gggggaggtg 60atctccgtcc acagcctgga gcagtggacc atgcagatcg aggaggccaa cgccgccaag 120aagctggtgg tgattgactt cactgcatca tggtgcggac catgccgcat tatggctcca 180attttcgctg atctcgccaa gaagttccca gctgctgttt tcctcaaggt cgacgttgat 240gaactgaagc ccattgctga gcaattcagc gtggaggcca tgccaacctt cctgttcatg 300aaggaaggag atgtcaagga cagggttgtc ggagctatca aggaggaact gacgaccaag 360gttgggctac acgcggcccc ag 382 4 127 PRT Triticum Aestivum 4 Met Ala AlaSer Ala Ala Thr Ala Thr Ala Thr Ala Ala Ala Val Gly 1 5 10 15 Arg GlyGlu Val Ile Ser Val His Ser Leu Glu Gln Trp Thr Met Gln 20 25 30 Ile GluGlu Ala Asn Ala Ala Lys Lys Leu Val Val Ile Asp Phe Thr 35 40 45 Ala SerTrp Cys Gly Pro Cys Arg Ile Met Ala Pro Ile Phe Ala Asp 50 55 60 Leu AlaLys Lys Phe Pro Ala Ala Val Phe Leu Lys Val Asp Val Asp 65 70 75 80 GluLeu Lys Pro Ile Ala Glu Gln Phe Ser Val Glu Ala Met Pro Thr 85 90 95 PheLeu Phe Met Lys Glu Gly Asp Val Lys Asp Arg Val Val Gly Ala 100 105 110Ile Lys Glu Glu Leu Thr Thr Lys Val Gly Leu His Ala Ala Gln 115 120 1255 393 DNA Artificial Sequence Wheat Thioredoxin cDNA 5 atggcggcggcggcgacggc gacgactaca gcggcggcga cggcggcggc ggtggggccg 60 ggggaggtgatctccgtcca cagcctggag cagtggacca tgcagatcga ggaggccaac 120 gccgccaagaagctggtggt gattgacttc actgcatcat ggtgcggacc atgccgcatt 180 atggctccaatttttgctga tctcgccaag aagttcccag ctgctgtttt cctcaaggtc 240 gacgttgatgaactgaagcc cattgctgag caattcagcg tcgaggccat gccaaccttc 300 ctgttcatgaaggaaggaga cgtcaaggac agggttgtcg gagctatcaa ggaggagctg 360 acgaccaaggttgggctcca cgcggctgcc tag 393 6 130 PRT Triticum Durum 6 Met Ala Ala AlaAla Thr Ala Thr Thr Thr Ala Ala Ala Thr Ala Ala 1 5 10 15 Ala Val GlyPro Gly Glu Val Ile Ser Val His Ser Leu Glu Gln Trp 20 25 30 Thr Met GlnIle Glu Glu Ala Asn Ala Ala Lys Lys Leu Val Val Ile 35 40 45 Asp Phe ThrAla Ser Trp Cys Gly Pro Cys Arg Ile Met Ala Pro Ile 50 55 60 Phe Ala AspLeu Ala Lys Lys Phe Pro Ala Ala Val Phe Leu Lys Val 65 70 75 80 Asp ValAsp Glu Leu Lys Pro Ile Ala Glu Gln Phe Ser Val Glu Ala 85 90 95 Met ProThr Phe Leu Phe Met Lys Glu Gly Asp Val Lys Asp Arg Val 100 105 110 ValGly Ala Ile Lys Glu Glu Leu Thr Thr Lys Val Gly Leu His Ala 115 120 125Ala Ala 130 7 26 DNA Artificial Sequence Primer 7 gtaaagcntt aacaacccacacattg 26 8 34 DNA Artificial Sequence Primer 8 ccgacgccgc tgcaatcgtacttgttgccg caat 34 9 332 PRT Hordeum Vulgare 9 Met Glu Gly Ser Ala AlaAla Pro Leu Arg Thr Arg Val Cys Ile Ile 1 5 10 15 Gly Ser Gly Pro AlaAla His Thr Ala Ala Ile Tyr Ala Ala Arg Ala 20 25 30 Glu Leu Lys Pro ValLeu Phe Glu Gly Trp Met Ala Asn Asp Ile Ala 35 40 45 Ala Gly Gly Gln LeuThr Thr Thr Thr Asp Val Glu Asn Phe Pro Gly 50 55 60 Phe Pro Thr Gly IleMet Gly Ile Asp Leu Met Asp Asn Cys Arg Ala 65 70 75 80 Gln Ser Val ArgPhe Gly Thr Asn Ile Leu Ser Glu Thr Val Thr Glu 85 90 95 Val Asp Phe SerAla Arg Pro Phe Arg Val Thr Ser Asp Ser Thr Thr 100 105 110 Val Leu AlaAsp Thr Val Val Val Ala Thr Gly Ala Val Ala Arg Arg 115 120 125 Leu HisPhe Ser Gly Ser Asp Thr Tyr Trp Asn Arg Gly Ile Ser Ala 130 135 140 CysAla Val Cys Asp Gly Ala Ala Pro Ile Phe Arg Asn Lys Pro Ile 145 150 155160 Ala Val Ile Gly Gly Gly Asp Ser Ala Met Glu Glu Gly Asn Phe Leu 165170 175 Thr Lys Tyr Gly Ser Gln Val Tyr Ile Ile His Arg Arg Asn Thr Phe180 185 190 Arg Ala Ser Lys Ile Met Gln Ala Arg Ala Leu Ser Asn Pro LysIle 195 200 205 Gln Val Val Trp Asp Ser Glu Val Val Glu Ala Tyr Gly GlyAla Gly 210 215 220 Gly Gly Pro Leu Ala Gly Val Lys Val Lys Asn Leu ValThr Gly Glu 225 230 235 240 Val Ser Asp Leu Gln Val Ser Gly Leu Phe PheAla Ile Gly His Glu 245 250 255 Pro Ala Thr Lys Phe Leu Asn Gly Gln LeuGlu Leu His Ala Asp Gly 260 265 270 Tyr Val Ala Thr Lys Pro Gly Ser ThrHis Thr Ser Val Glu Gly Val 275 280 285 Phe Ala Ala Gly Asp Val Gln AspLys Lys Tyr Arg Gln Ala Ile Thr 290 295 300 Ala Ala Gly Ser Gly Cys MetAla Ala Leu Asp Ala Glu His Tyr Leu 305 310 315 320 Gln Glu Val Gly AlaGln Val Gly Lys Ser Asp Glx 325 330 10 995 DNA Hordeum Vulgare 10atggagggat ccgccgcggc gccgctccgc acgcgcgtgt gcatcatcgg cagcggcccg 60gccgcgcaca cggcggccat ctacgcggcc cgcgcggagc tcaagcccgt gctcttcgag 120ggctggatgg ccaacgacat cgccgcgggg ggccagctca ccaccaccac cgacgtcgag 180aacttccccg gattccccac cggcatcatg ggcatcgacc tcatggacaa ctgccgcgcc 240cagtccgtcc gcttcggcac caacatcctc tccgagaccg tcaccgaggt cgacttctcc 300gcccgcccct tccgcgtcac ctccgactcc accaccgtcc tcgccgacac cgtcgtcgtc 360gccacgggcg ccgtcgcgcg ccgcctccat ttctccggtt ccgacaccta ctggaaccgc 420ggcatctccg cctgcgccgt ctgcgacggc gctgcgccca tcttccggaa caagcccatc 480gccgtcatcg gcggcggtga ttccgccatg gaggaaggca acttcctcac caagtacgga 540tcccaagtgt acatcatcca cgggcgcaac accttccgcg cctccaagat tatgcaggct 600agggcgctct ccaatcctaa gatccaggtt gtctgggact cgaggtcgtc gaggcttacg 660gcggtgcagg cggcggccca ttagctgggg tcaaggtcaa gaacttggtg actggtgagg 720tgtctgacct tcaggtgtcc gggcttttct tcgccatcgg gcatgagccg gccaccaagt 780ttctcaatgg gcagcttgag ctccatgccg atgggtatgt ggccaccaag ccgggctcta 840cacataccag tgtggagggg tctttgctgc tggagacgtg caggataaga agtatcgtca 900ggccattact gctgctggat caggttgcat ggctgctttg ggacgccgag cactatctgc 960aggaggtggg tgcacaggtg ggcaagtctg attga 995 11 486 DNA Barley 11aagctttaac aacccacaca ttgattgcaa cttagtccta cacaagtttt ccattcttgt 60ttcaggctaa caacctatac aaggttccaa aatcatgcaa aagtgatgct aggttgataa 120tgtgtgacat gtaaagtgaa taaggtgagt catgcatacc aaacctcggg atttctatac 180tttgtgtatg atcatatgca caactaaaag gcaactttga ttatcaattg aaaagtaccg 240cttgtagctt gtgcaaccta acacaatgtc caaaaatcca tttgcaaaag catccaaaca 300caattgttaa agctgttcaa acaaacaaag aagagatgaa gcctggctac tataaatagg 360caggtagtat agagatctac acaagcacaa gcatcaaaac caagaaacac tagttaacac 420caatccacta tgaagacctt cctcatcttt gcactcctcg ccattgcggc aacaagtacg 480attgca 486 12 497 DNA Barley 12 cttcgagtgc ccgccgattt gccagcaatggctaacagac acatattctg ccaaaacccc 60 agaacaataa tcacttctcg tagatgaagagaacagacca agatacaaac gtccacgctt 120 cagcaaacag taccccagaa ctaggattaagccgattacg cggctttagc agaccgtcca 180 aaaaaactgt tttgcaaagc tccaattcctccttgcttat ccaatttctt ttgtgttggc 240 aaactgcact tgtccaaccg attttgttcttcccgtgttt cttcttaggc taactaacac 300 agccgtgcac atagccatgg tccggaatcttcacctcgtc cctataaaag cccagccaat 360 ctccacaatc tcatcatcac cgagaacaccgagaaccaca aaactagaga tcaattcatt 420 gacagtccac cgagatggct aagcggctggtcctctttgt ggcggtaatc gtcgccctcg 480 tggctctcac caccgct 497 13 23 DNAArtificial Sequence Degenerative Primer 13 ttcttcgcsa tcggmcayga rcc 2314 23 DNA Artificial Sequence Degenerative Primer 14 gcgtcsarrgcrgccatgca scc 23 15 23 DNA Artificial Sequence Primer 15 acsacsacsacsgacgtsga raa 23 16 19 DNA Artificial Sequence Primer 16 actggtatgtgtagagccc 19 17 20 DNA Artificial Sequence Primer 17 aattaaccctcactaaaggg 20 18 21 DNA Artificial Sequence Primer 18 aagttctcgacgtcggtggt g 21 19 17 DNA Artificial Sequence Primer 19 caggaaacagctatgac 17 20 21 DNA Artificial Sequence Primer 20 attatgcagg ctagggcgctc 21 21 30 DNA Artificial Sequence Primer 21 tatctagaat ggagggatccgccgcggcgc 30 22 30 DNA Artificial Sequence Primer 22 ttggtacctcaatcagactt gcccacctgt 30 23 995 DNA Hordeum Vulgare 23 atggagggatccgccgcggc gccgctccgc acgcgcgtgt gcatcatcgg cagcggcccg 60 gccgcgcacacggcggccat ctacgcggcc cgcgcggagc tcaagcccgt gctcttcgag 120 ggctggatggccaacgacat cgccgcgggg ggccagctca ccaccaccac cgacgtcgag 180 aacttccccggattccccac cggcatcatg ggcatcgacc tcatggacaa ctgccgcgcc 240 cagtccgtccgcttcggcac caacatcctc tccgagaccg tcaccgaggt cgacttctcc 300 gcccgccccttccgcgtcac ctccgactcc accaccgtcc tcgccgacac cgtcgtcgtc 360 gccacgggcgccgtcgcgcg ccgcctccat ttctccggtt ccgacaccta ctggaaccgc 420 ggcatctccgcctgcgccgt ctgcgacggc gctgcgccca tcttccggaa caagcccatc 480 gccgtcatcggcggcggtga ttccgccatg gaggaaggca acttcctcac caagtacgga 540 tcccaagtgtacatcatcca cgggcgcaac accttccgcg cctccaagat tatgcaggct 600 agggcgctctccaatcctaa gatccaggtt gtctgggact cgaggtcgtc gaggcttacg 660 gcggtgcaggcggcggccca ttagctgggg tcaaggtcaa gaacttggtg actggtgagg 720 tgtctgaccttcaggtgtcc gggcttttct tcgccatcgg gcatgagccg gccaccaagt 780 ttctcaatgggcagcttgag ctccatgccg atgggtatgt ggccaccaag ccgggctcta 840 cacataccagtgtggagggg tctttgctgc tggagacgtg caggataaga agtatcgtca 900 ggccattactgctgctggat caggttgcat ggctgctttg ggacgccgag cactatctgc 960 aggaggtgggtgcacaggtg ggcaagtctg attga 995 24 332 PRT Arabidopsis Thaliana 24 MetAsn Gly Leu Glu Thr His Asn Thr Arg Leu Cys Ile Val Gly Ser 1 5 10 15Gly Pro Ala Ala His Thr Ala Ala Ile Tyr Ala Ala Arg Ala Glu Leu 20 25 30Lys Pro Leu Leu Phe Glu Gly Trp Met Ala Asn Asp Ile Ala Pro Gly 35 40 45Gly Gln Leu Asn Gln Pro Pro Arg Glu Asn Phe Pro Gly Phe Pro Glu 50 55 60Gly Ile Leu Gly Val Glu Leu Thr Asp Lys Phe Arg Lys Gln Ser Glu 65 70 7580 Arg Phe Gly Thr Thr Ile Phe Thr Glu Thr Val Thr Lys Val Asp Phe 85 9095 Ser Ser Lys Pro Phe Lys Leu Phe Thr Asp Ser Lys Ala Ile Leu Ala 100105 110 Asp Ala Val Ile Leu Ala Ile Gly Ala Val Ala Lys Trp Leu Ser Phe115 120 125 Val Gly Ser Gly Glu Val Leu Gly Gly Leu Trp Asn Arg Gly IleSer 130 135 140 Ala Cys Ala Val Cys Asp Gly Ala Ala Pro Ile Phe Arg AsnLys Pro 145 150 155 160 Leu Ala Val Ile Gly Gly Gly Asp Ser Ala Met GluGlu Ala Asn Phe 165 170 175 Leu Thr Lys Tyr Gly Ser Lys Val Tyr Ile IleAsp Arg Arg Asp Ala 180 185 190 Phe Arg Ala Ser Lys Ile Met Gln Gln ArgAla Leu Ser Asn Pro Lys 195 200 205 Ile Asp Val Ile Trp Asn Ser Ser ValVal Glu Ala Tyr Gly Asp Gly 210 215 220 Glu Arg Asp Val Leu Gly Gly LeuLys Val Lys Asn Val Val Thr Gly 225 230 235 240 Asp Val Ser Asp Leu LysVal Ser Gly Leu Phe Phe Ala Ile Gly His 245 250 255 Glu Pro Ala Thr LysPhe Leu Asp Gly Gly Val Glu Leu Asp Ser Asp 260 265 270 Gly Tyr Val ValThr Lys Pro Gly Thr Thr Gln Thr Ser Val Pro Gly 275 280 285 Val Phe AlaAla Gly Asp Val Gln Asp Lys Lys Tyr Arg Gln Ala Ile 290 295 300 Thr AlaAla Gly Thr Gly Cys Met Ala Ala Leu Asp Ala Glu His Tyr 305 310 315 320Leu Gln Glu Ile Gly Ser Gln Gln Gly Lys Ser Asp 325 330 25 321 PRTEscherichia Coli 25 Met Gly Thr Thr Lys His Ser Lys Leu Leu Ile Leu GlySer Gly Pro 1 5 10 15 Ala Gly Tyr Thr Ala Ala Val Tyr Ala Ala Arg AlaAsn Leu Gln Pro 20 25 30 Val Leu Ile Thr Gly Met Glu Lys Gly Gly Gln LeuThr Thr Thr Thr 35 40 45 Glu Val Glu Asn Trp Pro Gly Asp Pro Asn Asp LeuThr Gly Pro Leu 50 55 60 Leu Met Glu Arg Met His Glu His Ala Thr Lys PheGlu Thr Glu Ile 65 70 75 80 Ile Phe Asp His Ile Asn Lys Val Asp Leu GlnAsn Arg Pro Phe Arg 85 90 95 Leu Asn Gly Asp Asn Gly Glu Tyr Thr Cys AspAla Leu Ile Ile Ala 100 105 110 Thr Gly Ala Ser Ala Arg Tyr Leu Gly LeuPro Ser Glu Glu Ala Phe 115 120 125 Lys Gly Arg Gly Val Ser Ala Cys AlaThr Cys Asp Gly Phe Phe Tyr 130 135 140 Arg Asn Gln Lys Val Ala Val IleGly Gly Gly Asn Thr Ala Val Glu 145 150 155 160 Glu Ala Leu Tyr Leu SerAsn Ile Ala Ser Glu Val His Leu Ile His 165 170 175 Arg Arg Asp Gly PheArg Ala Glu Lys Ile Leu Ile Lys Arg Leu Met 180 185 190 Asp Lys Val GluAsn Gly Asn Ile Ile Leu His Thr Asn Arg Thr Leu 195 200 205 Glu Glu ValThr Gly Asp Gln Met Gly Val Thr Gly Val Arg Leu Arg 210 215 220 Asp ThrGln Asn Ser Asp Asn Ile Glu Ser Leu Asp Val Ala Gly Leu 225 230 235 240Phe Val Ala Ile Gly His Ser Pro Asn Thr Ala Ile Phe Glu Gly Gln 245 250255 Leu Glu Leu Glu Asn Gly Tyr Ile Lys Val Gln Ser Gly Ile His Gly 260265 270 Asn Ala Thr Gln Thr Ser Ile Pro Gly Val Phe Ala Ala Gly Asp Val275 280 285 Met Asp His Ile Tyr Arg Gln Ala Ile Thr Ser Ala Gly Thr GlyCys 290 295 300 Met Ala Ala Leu Asp Ala Glu Arg Tyr Leu Asp Gly Leu AlaAsp Ala 305 310 315 320 Lys 26 998 DNA Arabidopsis thaliana 26atgaatggtc tcgaaactca caacacaagg ctctgtatcg taggaagtgg cccagcggca 60cacacggcgg cgatttacgc agctagggct gaacttaaac ctcttctctt cgaaggatgg 120atggctaacg acatcgctcc cggtggtcaa ctcaaccaac caccgcgtga gaatttcccc 180ggatttccag aaggtattct cggagtagag ctcactgaca aattccgtaa acaatcggag 240cgattcggta ctacgatatt tacagagacg gtgacgaaag tcgatttctc ttcgaaaccg 300tttaagctat tcacagattc aaaagccatt ctcgctgacg ctgtgattct cgctatcgga 360gctgtggcta agtggcttag cttcgttgga tctggtgaag ttctcggagg tttgtggaac 420cgtggaatct ccgcttgtgc tgtttgcgac ggagctgctc cgatattccg caacaaacct 480cttgcggtga tcggtggagg cgattctgca atggaagaag caaactttct tacaaaatat 540ggatctaaag tgtatataat cgataggaga gatgctttta gagcgtctaa gattatgcag 600cagcgacttt gtctaatcct aagattgatg tgatttggaa ctcgtctgtt gtggaagctt 660atggagatgg agaaagagat gtgcttggag gattgaaagt gaagaatgtg gttaccggag 720atgtttctga tttaaaagtt tctggattgt tctttgctat tggtcatgag ccagctacca 780agtttttgga tggtggtgtt gagttagatt cggatggtta tgttgtcacg aagcctggta 840ctacacagac tagcgttccc ggagttttcg ctgcgggtga tgttcaggat aagaagtata 900ggcaagccat cactgctgca ggaactgggt gcatggcagc tttggatgca gagcattact 960tacaagagat tggatctcag caaggtaaga gtgattga 998 27 966 DNA EscherichiaColi 27 atgggcacga ccaaacacag taaactgctt atcctgggtt caggcccggcgggatacacc 60 gctgctgtct acgcggcgcg cgccaacctg caacctgtgc tgattaccggcatggaaaaa 120 ggcggccaac tgaccaccac cacggaagtg gaaaactggc ctggcgatccaaacgatctg 180 accggtccgt tattaatgga gcgcatgcac gaacatgcca ccaagtttgaaactgagatc 240 atttttgatc atatcaacaa ggtggatctg caaaaccgtc cgttccgtctgaatggcgat 300 aacggcgaat acacttgcga cgcgctgatt attgccaccg gagcttctgcacgctatctc 360 ggcctgccct ctgaagaagc ctttaaaggc cgtggggttt ctgcttgtgcaacctgcgac 420 ggtttcttct atcgcaacca gaaagttgcg gtcatcggcg gcggcaataccgcggttgaa 480 gaggcgctgt atctgtctaa catcgcttcg gaagtgcatc tgattcaccgccgtgacggt 540 ttccgcgcgg aaaaaatcct cattaagcgc ctgatggata aagtggagaacggcaacatc 600 attctgcaca ccaaccgtac gctggaagaa gtgaccggcg atcaaatgggtgtcactggc 660 gttcgtctgc gcgatacgca aaacagcgat aacatcgagt cactcgacgttgccggtctg 720 tttgttgcta tcggtcacag cccgaatact gcgattttcg aagggcagctggaactggaa 780 aacggctaca tcaaagtaca gtcgggtatt catggtaatg ccacccagaccagcattcct 840 ggcgtctttg ccgcaggcga cgtgatggat cacatttatc gccaggccattacttcggcc 900 ggtacaggct gcatggcagc acttgatgcg gaacgctacc tcgatggtttagctgacgca 960 aaataa 966 28 28 DNA Artificial Sequence Primer 28atatctagaa tggcggcgtc ggcggcga 28 29 27 DNA Artificial Sequence Primer29 atagagctct tactgggccg cgtgtag 27 30 36 DNA Artificial Sequence Primer30 ggcgcatgcg aattcgaatt cgatatcgat cttcga 36 31 27 DNA ArtificialSequence Primer 31 aactctagac tcggtggact gtcaatg 27 32 19 DNA ArtificialSequence Primer 32 ccaagaagtt cccagctgc 19 33 23 DNA Artificial SequencePrimer 33 atagctgcga caaccctgtc ctt 23 34 25 DNA Artificial SequencePrimer 34 catcgagaca agcacggtca acttc 25 35 24 DNA Artificial SequencePrimer 35 atatccgagc gcctcgtgca tgcg 24 36 27 PRT Rattus Rattus 36 TyrMet Thr Val Ser Ile Ile Asp Arg Phe Met Gln Asp Ser Cys Val 1 5 10 15Pro Lys Lys Met Leu Gln Leu Val Gly Val Thr 20 25 37 28 PRT Mus Musculus37 Lys Phe Arg Leu Leu Gln Glu Thr Met Tyr Met Thr Val Ser Ile Ile 1 510 15 Asp Arg Phe Met Gln Asn Ser Cys Val Pro Lys Lys 20 25 38 27 PRTMus Musculus 38 Arg Ala Ile Leu Ile Asp Trp Leu Ile Gln Val Gln Met LysPhe Arg 1 5 10 15 Leu Leu Gln Glu Thr Met Tyr Met Thr Val Ser 20 25 3927 PRT Mus Musculus 39 Asp Arg Phe Leu Gln Ala Gln Leu Val Cys Arg LysLys Leu Gln Val 1 5 10 15 Val Gly Ile Thr Ala Leu Leu Leu Ala Ser Lys 2025 40 18 PRT Mus Musculus 40 Met Ser Val Leu Arg Gly Lys Leu Gln Leu ValGly Thr Ala Ala Met 1 5 10 15 Leu Leu 41 26 DNA Artificial SequencePrimer 41 gtaaagcttt aacaacccac acattg 26 42 42 DNA Artificial SequencePrimer 42 cgccgttgcc gacgccgctg caatcgtact tgttgccgca at 42 43 32 DNAArtificial Sequence Primer 43 acaagtacga ttgcagcggc gtcggcaacg gc 32 4427 DNA Artificial Sequence Primer 44 atagagctct tactgggccg ccgcgtg 27 4519 DNA Artificial Sequence Primer 45 ccaagaagtt cccaaatgc 19 46 35 DNAArtificial Sequence Primer 46 attctagaat ggagggatcc gccgcggcgc cgctc 3547 30 DNA Artificial Sequence Primer 47 ttggtacctc aatcagactt gcccacctgt30 48 9 PRT Artificial Sequence Cyclin A Destruction Box 48 Arg Thr ValLeu Gly Val Ile Gly Asp 1 5 49 19 DNA Artificial Sequence Primer 49ccaagaagtt cccagcgtc 19 50 18 DNA Artificial Sequence Primer 50cacgcggcgg cccagtaa 18 51 9 PRT Artificial Sequence Cyclin B1Destruction Box 51 Arg Thr Ala Leu Gly Asp Ile Gly Asn 1 5

We claim:
 1. A recombinant nucleic acid encoding an NTR protein thatcatalyzes the reduction of thioredoxin coupled to NADPH₂ oxidationcomprising a nucleic acid that hybridizes to SEQ ID NO:10 underhybridization conditions that include at least one wash in 0.1×SSC at65° C.
 2. The recombinant nucleic acid of claim 1 comprising SEQ IDNO:10.
 3. A recombinant nucleic acid encoding an NTR protein thatcatalyzes the reduction of thioredoxin coupled to NADPH₂ oxidationcomprising a nucleic acid having at least 95% sequence identity to SEQID NO:10.
 4. A recombinant nucleic acid encoding SEQ ID NO:9.
 5. A hostcell comprising the recombinant nucleic acid of claim
 1. 6. Anexpression vector comprising the recombinant nucleic acid of claim 1operably linked to a transcriptional regulatory sequence.
 7. A host cellcomprising an expression vector comprising the recombinant nucleic acidof claim 1 operably linked to a transcriptional regulatory sequenceactive in said host cell.
 8. A transgenic plant comprising a recombinantnucleic acid encoding an NTR protein that catalyzes the reduction ofthioredoxin coupled to NADPH₂ oxidation comprising a nucleic acid thathybridizes to SEQ ID NO:10 under hybridization conditions that includeat least one wash in 0.1×SSC at 65° C.
 9. A transgenic plant of claim 8wherein said recombinant nucleic acid is operably linked to atranscriptional regulatory sequence active in said plant.
 10. Thetransgenic plant of claim 9 wherein said transcriptional regulatorysequence is active in a seed cell.
 11. A transgenic seed comprising atranscriptional regulatory sequence active in said seed operably linkedto a recombinant nucleic acid encoding an NTR protein that catalyzes thereduction of thioredoxin coupled to NADPH₂ oxidation comprising anucleic acid that hybridizes to SEQ ID NO:10 under hybridizationconditions that include at least one wash in 0.1×SSC at 65° C.
 12. Amethod of expressing an NTR protein comprising culturing a host cellcomprising the recombinant nucleic acid of claim 1 under conditionssuitable for expression of said NTR protein.
 13. A method of expressingan NTR protein comprising culturing a host cell comprising an expressionvector comprising the recombinant nucleic acid of claim 1 operablylinked to regulatory sequences active in said host cell under conditionssuitable for expression of said NTR protein.
 14. A method of expressingan NTR protein comprising culturing the transgenic plant of claim 8under conditions suitable for expression of said NTR protein.
 15. Amethod of expressing an NTR protein comprising culturing the transgenicplant of claim 9 under conditions suitable for expression of said NTRprotein.
 16. A method of expressing an NTR protein comprising culturingthe transgenic seed of claim 11 under conditions suitable for expressionof said NTR protein.
 17. The method of claim 12 further comprisingrecovering said protein.
 18. An isolated nucleic acid encoding an NTRprotein that catalyzes the reduction of thioredoxin coupled to NADPH₂oxidation comprising a nucleic acid that hybridizes to SEQ ID NO:10under hybridization conditions that include at least one wash in 0.1×SSCat 65° C.
 19. The isolated nucleic acid of claim 18 comprising SEQ IDNO:10.
 20. An isolated nucleic acid encoding an NTR protein thatcatalyzes the reduction of thioredoxin coupled to NADPH₂ oxidationcomprising a nucleic acid having at least 95% sequence identity to SEQID NO:10.
 21. An isolated nucleic acid encoding SEQ ID NO:9.
 22. Anexpression vector comprising the recombinant nucleic of claim 2 operablylinked to a transcriptional regulatory sequence.
 23. An expressionvector comprising the recombinant nucleic of claim 3 operably linked toa transcriptional regulatory sequence.
 24. An expression vectorcomprising the recombinant nucleic of claim 4 operably linked to atranscriptional regulatory sequence.
 25. A host cell comprising anexpression vector comprising the recombinant nucleic acid of claim 2operably linked to a transcriptional regulatory sequence active in saidhost cell.
 26. A host cell comprising an expression vector comprisingthe recombinant nucleic acid of claim 3 operably linked to atranscriptional regulatory sequence active in said host cell.
 27. A hostcell comprising an expression vector comprising the recombinant nucleicacid of claim 4 operably linked to a transcriptional regulatory sequenceactive in said host cell.
 28. A transgenic plant comprising arecombinant nucleic acid comprising SEQ ID NO:10.
 29. A transgenic plantcomprising a recombinant nucleic acid encoding an NTR protein thatcatalyzes the reduction of thioredoxin coupled to NADPH₂ oxidationcomprising a nucleic acid having at least 95% sequence identity to SEQID NO:10.
 30. A transgenic plant comprising a recombinant nucleic acidencoding SEQ ID NO:9.
 31. The transgenic plant of claim 28 wherein therecombinant nucleic acid is operably linked to a transcriptionalregulatory sequence active in said plant.
 32. The transgenic plant ofclaim 29 wherein the recombinant nucleic acid is operably linked to atranscriptional regulatory sequence active in said plant.
 33. Thetransgenic plant of claim 30 wherein the recombinant nucleic acid isoperably linked to a transcriptional regulatory sequence active in saidplant.
 34. A transgenic plant comprising a host cell comprising anexpression vector comprising a transcriptional regulatory sequenceactive in said cell operably linked to a recombinant nucleic acidcomprising SEQ ID NO:10.
 35. A transgenic plant comprising a host cellcomprising an expression vector comprising a transcriptional regulatorysequence active in said cell operably linked to a recombinant nucleicacid encoding SEQ ID NO:9.
 36. A transgenic seed comprising atranscriptional regulatory sequence active in said seed operably linkedto a recombinant nucleic acid comprising SEQ ID NO:10.
 37. A transgenicseed comprising a transcriptional regulatory sequence active in saidseed operably linked to a recombinant nucleic acid encoding an NTRprotein that catalyzes the reduction of thioredoxin coupled to NADPH₂oxidation comprising a nucleic acid having at least 95% sequenceidentity to SEQ ID NO:10.
 38. A transgenic seed comprising atranscriptional regulatory sequence active in said seed operably linkedto a recombinant nucleic acid.
 39. The method of claim 13 furthercomprising recovering said protein.
 40. The method of claim 14 furthercomprising recovering said protein.
 41. The method of claim 15 furthercomprising recovering said protein.
 42. The method of claim 16 furthercomprising recovering said protein.